Journal: Nature

Article Title: Chaperone-mediated autophagy sustains hematopoietic stem cell function

doi: 10.1038/s41586-020-03129-z

Figure Lengend Snippet: a-c, Percentage of stem cells in active cell cycle (not in G 0 by Ki67 and Hoechst staining) ( a ), ATP levels ( b ) and median fluorescence intensity (MFI) for cellular ROS (reactive oxygen species) in HSC from control (Ctrl) and L2AKO mice untreated (basal) or at day 8 after a single 5FU injection. n= 6–7 mice ( a ), 4–5 mice ( b ), 5–15 mice. Representative examples of FACS plots for and cycling analysis by Brdu incorporation are shown in - . d, ROS levels in Ctrl and L2AKO bone marrow (BM) derived HSC in transplanted recipients. n= 5 mice . e,f, Heat map of gene expression ( e ) and enrichment pathway analysis ( f ) in HSC cells from Ctrl and L2AKO mice untreated or 8 days post 5FU injection. g, STRING analysis of proteins whose levels decrease in control cells upon activation (top) and those at higher levels in L2AKO LSK than Ctrl LSK 8 days post-5FU (bottom). h, Number of proteins different and overlapping in the same two groups as in g . n=3 proteomic experiments with pool of 3 mice per group. i-k, Metabolic phenotypes of LSK cells from Ctrl and L2AKO mice. Unsupervised principal component analysis of the two groups ( i ), hierarchical clustering analysis of the top 25 significant metabolites by two-tailed t test ( j ) and Omicsnet analysis of the most significantly affected pathways in L2AKO cells ( k ). n=3 metabolomic experiments with pool of 3 mice per group. l , Extracellular acidification rates (ECAR) in Ctrl and L2AKO LSK cells and changes upon addition of glucose (Glu), oligomycin (Oligo) and 2-Deoxy-D-glucose (2DG). n=3 independent experiments. m, Basal glycolysis (left), glycolytic capacity (middle) and glycolytic reserve (right) in L2AKO LSK cells relative to control cells. n=3 independent experiments. n. GAPDH (left) and pyruvate kinase (PK) (right) activity in Ctrl and L2AKO LSK cells. n=3 independent experiments. o, Percentage of cellular oxidized GAPDH and PK detected by mass spec in Ctrl and L2AKO LSK cells under basal conditions. n=9 mice in 3 different experiments. p, Percentage of total cellular proteins that are oxidized (left) and fold changes in the number of carbonylated peptides (right) in LSK from Ctrl and L2AKO mice under basal conditions or 8 days after 5FU injection. n=9 mice in 3 independent experiments. q, r Oxidized proteins ( q ) and protein inclusions ( r ) in basal Ctrl and L2AKO HSC detected by staining with OxylCC and Proteostat, respectively. Representative images (left) and quantification of staining intensity (right) are shown. n=15 fields from 5 individual mice (individual points represent the average of 5–10 cells per field). Nuclei are highlighted with DAPI. Full field images for q and r as shown in . Two-way ANOVA test followed by Sidak’s ( a-c ) or Tukey’s ( p ) multiple comparison post-hoc test, Chi-square test ( k ), multiple time point paired t-test ( l ) and unpaired t-test ( d,m-o,q, r ) were used for the statistics. P<0.05 (*), 0.01 (**), 0.001(***), 0.0001 (****). ns: no statistical significance.

Article Snippet: Mono-nuclear cells were further enriched for CD34 + cells using immunomagnetic bead sorting (CD34 MicroBead Kit, human; Miltenyi Biotec) according to the manufacturer’s protocol.

Techniques: Staining, Fluorescence, Control, Injection, BrdU Incorporation Assay, Derivative Assay, Gene Expression, Activation Assay, Two Tailed Test, Activity Assay, Mass Spectrometry, Comparison

Journal: Nature

Article Title: Chaperone-mediated autophagy sustains hematopoietic stem cell function

doi: 10.1038/s41586-020-03129-z

Figure Lengend Snippet: a, Top lipid metabolism pathways identified from enrichment analysis of metabolites as different between control and L2AKO LSK cells 8 days post 5FU injection. b, Heat map of substrate and metabolite abundance in the linoleic acid and α-linolenic metabolism in the indicated conditions. c, Scheme of linoleic and α-linolenic fatty acid metabolism pathway showing the increased (red arrow) and decreased (green arrow) metabolites in L2AKO LSK cells comparing with Ctrl cells in 5FU-activated conditions. d, Levels of substrates (black, gray) and downstream metabolites (green) of the linoleic acid and α-linolenic metabolism relative to those in unstimulated control cells. n= 9 mice in 3 different experiments. Significant differences with control untreated are marked with * and between L2AKO basal and 5FU treated with †. e, Total colony numbers (left) and number of immature, multipotent colony-forming unit-granulocyte/ erythroid/macrophage/megakaryocyte CFU-GEMM (right) in the first plating from Ctrl and L2AKO HSC treated or not with the FADS2 inhibitor SC-26196 (SC). n=3 independent experiments. f, Total colony numbers in the 3 rd plating (left) or number of CFU-GEMM colonies in the first plating (right) from Ctrl and L2AKO HSC treated or not with γ-linolenic acid (GLA). n=4 independent experiments. g, Predicted CMA-targeting motif in FADS2 (green) generated by acetylation of 42 K ( g top) and analysis of the acetylation level of this peptide detected by mass spectrometry Ctrl and L2AKO LSK cells in basal and 5FU activated conditions ( g bottom). n=3 mice. h,i, Immunoblot for Flag of Ctrl ( h ) and L2AKO ( i ) ex vivo expanded HSC expressing Flag-myc tagged FADS2 wild type (VIDRK) or mutated as indicated at the top (VIDRQ or VIDRA) treated or not with NH 4 Cl/Leupeptin (N/L). For gel source data, see . j, Oxygen consumption rate (OCR) in Ctrl and L2AKO LSK cells at day 8 after 5FU injection. Responses to addition of oligomycin (Oligo), FCCP and rotenone (Roteno) are shown. n=5 independent experiments. k, Quantification of mitochondrial respiration from fatty acid β-oxidation (etomoxir-sensitive) in Ctrl and L2AKO LSK cells. n=5 independent experiments. l , Total colony numbers in 3 rd plating from both Ctrl and L2AKO HSC treated or not with Methyl-pyruvate (5mM) starting from the 1 st plating. n=7 (None) and 4 (Methyl-pyruvate) independent mice. m, Quantification of MFI of ROS + cells (left) and total colony numbers (right) in the 3 rd plating of Ctrl or L2AKO HSC untreated or supplemented with NAC from the 1 st plating. n=7 (None) and 4 (NAC) mice. n, Ratio of γ-linolenic acid (GLA) and linoleic acid (LA) in LSK cells from young and old mice calculated from the metabolomics data. n= 9 mice in 3 different experiments. o, Ratio of K42 acetylated and total FADS2 peptide in LSK from young and old mice calculated from the mass spectrometry analysis. n= 9 mice in 3 different experiments. p, Total colony numbers (left) and number of CFU-GEMM colonies (right) in the colony formation assay with HSC cells from old (22m) mice daily injected with either saline or GLA (1mg/kg bw) for 7 weeks. Representative images of wells with Ctrl or GLA treated cells are shown on the top. n=5 mice per group. q, LTC-IC assay according to GEMM colonies from Ctrl and GLA treated human CD34 + cells from old (>65 year) multi-myeloma patients. n=2 patients. Two-way ANOVA test followed by Tukey’s ( d,f ) or Sidak ( e,l,m ) multiple comparison post-hoc test, unpaired t-tests ( g,k,n,o,p ), time point paired t-test ( j ), and Chi-square test ( q ) were used for the statistics. P<0.05 (*), 0.01 (**), 0.001(***), 0.0001(****). ns: no statistical significance.

Article Snippet: Mono-nuclear cells were further enriched for CD34 + cells using immunomagnetic bead sorting (CD34 MicroBead Kit, human; Miltenyi Biotec) according to the manufacturer’s protocol.

Techniques: Control, Injection, Generated, Mass Spectrometry, Western Blot, Ex Vivo, Expressing, Colony Assay, Saline, Comparison

Journal: Nature

Article Title: Chaperone-mediated autophagy sustains hematopoietic stem cell function

doi: 10.1038/s41586-020-03129-z

Figure Lengend Snippet: a, Changes in HSC numbers per femur and tibia in Ctrl and L2AKO mice with age. Values are relative to young control mice. n=9–11 mice. b, Donor chimerism at the indicated time points in peripheral blood of recipients after competitive transplantation of 25–30m Ctrl or L2AKO BM cells. n=5–6 mice. c, Donor lineage distribution in peripheral blood 24 weeks after second competitive transplantation of Ctrl or L2AKO BM cells. n=5–6 mice. d, ROS levels in HSC from 3–4m and >25m old Ctrl and L2AKO mice. Data is shown as median fluorescence intensity (MFI) per cell. n=5–6 mice. e, Percentage of HSC in BM of young, old control and old mice bearing an extra copy of human L2A (hL2A OE ). n=5 (young), 18 (old ctrl) and 13 (old hL2A OE ) mice. f,g, Representative FACS ( f ) and quantification ( g ) of ROS levels in HSC cells from young, old Ctrl and hL2A OE mice. Data is shown relative to old Ctrl mice. n=5 mice. h-j PK ( h ) and GAPDH ( i ) activity and extracellular acidification rates (ECAR) in basal conditions or after addition of oligomycin (Oligo) and 2-deoxy-D-glucose (2DG) ( j ) in LSK cells from old Ctrl and hL2A OE mice. n=3 mice. k. Levels of polyunsaturated fatty acids generated by FADS2 in old Ctrl and hL2A OE mice expressed as peak area relative to young mice. n= 9 (young), 18 (old), 9 (hL2AOE) mice in 3 independent experiments. l. Percentage of donor derived cells at the indicated times in mice transplanted with BM from old Ctrl and hL2A OE mice. n=5 mice. m-o, Levels of oxidized proteins ( m ), GAPDH activity ( n ) and ECAR in basal conditions or upon Oligo and 2DG addition ( o ) in HSC from old (>22m) mice 2 months after daily oral administration of a chemical activator of CMA (CA 20mg/Kg b.w.) or the corresponding vehicle (Veh.). Values in young mice are shown as reference in n. Representative images of cells used for quantification in m are in . n=12 fields from 4 and 3 mice for m and n , respectively. p. LTC-IC assay of LSK cells from BM of old mice administered daily for 2 months CA or vehicle. Scatter plot shows LTC-IC frequency for vehicle and CA treated old mice. n=4 mice. q,r LTC-IC assay of old LSK cells after ex vivo treatment with vehicle (DMSO) or a chemical activator of CMA (CA 10μM, daily for 4 weeks) ( q ) and viable cell percentage recovered from the colonies formed at the end of LTC-IC ( r ). Individual plot showing the fold change of LTC-IC frequency compared to old control cells ( q left), scatter plot showing LTC-IC frequency for vehicle and CA treated old Ctrl cells ( q right). n=3 mice. s. Representative images of Giemsa staining of the cells recovered at the end of the LTC-IC assay when cells were plated in presence of CA (10μM) or vehicle. n=3 experiments. t,u. Quantification of immature GEMM colonies in the 1 st plating ( t ) and total colony numbers in the 2 nd plating ( u ) of CD34 + enriched stem and progenitor cells from mobilized blood of multi-myeloma patients (59, 65, and 71 years old), when maintained in presence of CA (10μM) or vehicle starting from the 1 st plating. n=3 patients. Multiple t-tests ( a-c ), unpaired t-tests ( m,q left ,r,t,u ), time points paired t-test ( j, o ), two-way ANOVA test followed by Sidak’s multiple comparisons post-hoc test ( d, l ), one-way ANOVA tests followed by Tukey’s ( e,g-i,n ), Sidak’s ( k ) or Dunnett’s ( n ) multiple comparisons post-hoc test and Chi-square test ( p,q right) were used for the statistics. P<0.05 (*), 0.01 (**), 0.001 (***), 0.001(****). ns: no statistical significance.

Article Snippet: Mono-nuclear cells were further enriched for CD34 + cells using immunomagnetic bead sorting (CD34 MicroBead Kit, human; Miltenyi Biotec) according to the manufacturer’s protocol.

Techniques: Control, Transplantation Assay, Fluorescence, Activity Assay, Generated, Derivative Assay, Ex Vivo, Staining

Journal: Nature genetics

Article Title: BRD2 Compartmentalizes the Accessible Genome

doi: 10.1038/s41588-022-01044-9

Figure Lengend Snippet: (a) Enrichment profile and heatmap of BRD2/3/4, CTCF, P300 ChIP-seq signal at their respective binding peaks before and after BRD4 depletion. (b) Representative genomic tracks (from integrated genomics viewer) of ChIP-seq signal of BRD2, BRD4 or CTCF after acute depletion of BRD4 for 6 hours. (c) Representative single cell view of Cohesin (RAD21, green), BRD2 (cyan) and BRD4 (magenta) and merged BRD2/BRD4. The cyan arrow indicates the BRD2 puncta showing little colocalization with BRD4. The magenta arrow indicates the BRD4 puncta poorly colocalized with BRD2. The white line indicates the region to profile the fluorescent intensity. Scale bar, 1μm. (d) Fluorescence intensity profile of BRD2(cyan) and BRD4 (magenta) along the while line in (c). The relative intensity ratio is plotted. (e) WB analysis of the Rad21-AID : Brd4-dTAG : Brd2-dTAG triple edited ESC line. dTAG13 treatment (100nM, 6 hours) simultaneously depletes both BRD2 and BRD4 whereas auxin treatment (100μM, 6 hours) orthogonally depletes RAD21. (f) Histogram of eigenvector values from the Pearson’s correlation matrix of single RAD21 depletion, dual RAD21/BRD4 or triple RAD21/BRD4/BRD2 depletion for 6 hours compared to untreated Control from Micro-C experiments. (g) Quantification of the digitalized A-A compartmental interactions (log10 value of observed/expected) after single RAD21 depletion, dual RAD21/BRD4 depletion or triple RAD21/BRD2/BRD4 depletion for 6 hours from Micro-C experiments. Triple RAD21/BRD4/BRD2 depletion reduced the enhanced compartmentalization after the dual Cohesin/BRD4 depletion. The black solid line in each violin plot represents the median value. (h) Degradation of BRD2 significantly reduced the accessible chromatin clustering after dual BRD4/RAD21 depletion. g(r) curves were plotted for indicated conditions. The non-parametric two-sided Mann-Whitney U test was used for statistical testing. (i) Biallelic knock-in of HaloTag into endogenous BET family genes enable accurate quantification of individual BET family protein copy number by CTCF-calibrated flow cytometry in live cells. The mean and standard deviation of the quantified copy number from two biological experiments are shown above each plot. The non-parametric Mann-Whitney U test was used for statistical testing. ***, p < 0.001; n.s., not significant.

Article Snippet: The full length (FL), N-terminus double bromodomain (BD, 1-508aa) and C-terminus region containing large segments of low complexity domains (LCDs, 447-798aa) of mouse BRD2 were PCR amplified from the RSV-Flag-mBrd2 vector (Addgene, 86614) and subcloned into a PiggyBac vector containing an N-terminus V5-SNAP tag.

Techniques: ChIP-sequencing, Binding Assay, Fluorescence, Control, MANN-WHITNEY, Knock-In, Flow Cytometry, Standard Deviation

Journal: Nature genetics

Article Title: BRD2 Compartmentalizes the Accessible Genome

doi: 10.1038/s41588-022-01044-9

Figure Lengend Snippet: (a) Low-dose 1,6 Hexanediol (1,6-HD,2%) treatment for 1.5 hours with or without Cohesin depletion did not significantly affect chromatin accessibility in the linear genome. The heatmaps of ATAC-seq enrichment at CTCF sites, enhancers and promoters are shown for WT, Cohesin depletion, 1,6 HD treatment, or a combination of both. (b) Disrupting BET-chromatin interactions by JQ1 had no significant impact on accessible chromatin clustering after CTCF depletion. g(r) curves were plotted for indicated conditions. Specifically, 1μM JQ1 treatment for 12 hours was applied to both control and CTCF depleted cells (6-hour depletion). The two-sided Mann-Whitney U test was used for statistical testing. (c-d) Violin plot of 3D volumes (the number of voxels) of two ATAC-rich segments R2 ( c ) and R5 ( d ) before (grey) and after (red) auxin-mediated CTCF depletion with or without JQ1 or 1,6HD treatment. The number of alleles analyzed is indicated at the bottom. The black bar indicates the median value for each data set and Mann-Whitney U test was performed for the statistical analysis. (e ) The disordered region predicted from PONDR program (Predictor of Natural Disordered Regions) for BET family proteins using the VSL2 algorithms. Except for the double bromodomain and the extra-terminal domain, other regions of BET proteins are predicted to be highly disordered with high disorder score. (f ) RNA-seq analysis of the expression level of BET family genes in mouse ESCs and mouse embryonic fibroblasts (MEF). Except for the Brdt gene, Brd2/3/4 are all actively transcribed in ESCs. RNA-seq data was derived from GSM723776 and GSM723775.

Article Snippet: The full length (FL), N-terminus double bromodomain (BD, 1-508aa) and C-terminus region containing large segments of low complexity domains (LCDs, 447-798aa) of mouse BRD2 were PCR amplified from the RSV-Flag-mBrd2 vector (Addgene, 86614) and subcloned into a PiggyBac vector containing an N-terminus V5-SNAP tag.

Techniques: Control, MANN-WHITNEY, RNA Sequencing, Expressing, Derivative Assay

Journal: Nature genetics

Article Title: BRD2 Compartmentalizes the Accessible Genome

doi: 10.1038/s41588-022-01044-9

Figure Lengend Snippet: (a) A schematic of the dual targeted protein degradation strategy. In Rad21-eGFP-mAID cells, a FKBP F36V based degron (dTAG) linked to a HaloTag was bi-allelically knocked into endogenous BET family genes ( Brd2, Brd3 and Brd4 ) by the CRISPR/Cas9 genome editing method (only one engineered allele is shown). Adding cell membrane permeable dTAG13 ligand into the culture will bring the dTAG labeled BET proteins into close proximity to the Cereblon (CRBN) E3 ligase for proteasome-mediated protein degradation orthogonal to the mAID system. (b) Western blot (WB) analysis of protein levels of endogenous BRD2-dTAG or RAD21-AID individually or in combination after 6 hours of dTAG13 or auxin treatment, respectively. Rapid depletion of BRD2 or RAD21 individually or together does not impact the protein level of other BET proteins (BRD3 or BRD4). (c) Degradation of BRD2 for 6 hours significantly reduced the accessible chromatin clustering after RAD21 depletion. g(r) curves were plotted for indicated conditions. (d-e) Violin plot of 3D volumes (the number of voxels) of two ATAC-rich segments R2 ( d ) and R5 ( e ) before and after BRD2 depletion, RAD21 depletion or in combination. The black bar indicates the median value for each data set and Mann-Whitney U test was performed for the statistical analysis. The number of analyzed alleles for the ATAC-rich segment (Chr4-R2) are : Control (n=93), RAD21 depletion (n=129), BRD2 depletion (n=116), or dual depletion of both RAD21 and BRD2 (n=119). The number of analyzed alleles for the ATAC-rich segment (Chr4-R5) are : Control (n=91), RAD21 depletion (n= 85), BRD2 depletion (n=99), or dual depletion of both RAD21 and BRD2 (n=121). (f) Pearson’s correlation matrix of the whole chromosome 17 for BRD2 or RAD21 depletion alone or in combination for 6 hours from Micro-C experiments. Dual BRD2 and RAD21 depletion reduced the enhanced compartmentalization after Cohesin depletion alone. (g ) Quantification of the digitalized A-A compartmental interactions (log10 value of observed/expected) after BRD2 or RAD21 depletion alone or in combination from Micro-C experiments. The black solid line in each violin plot represents the median value. (h-i) Differential saddle plot analysis (h) and quantitative A-A compartmental interactions (log10 value of observed/expected) analysis (i) by Micro-C for cells stably expressing empty vector, full length (FL), N-terminal double bromodomain (BD) and C-terminal low complexity domain (LCD) of BRD2. The non-parametric two-sided Mann-Whitney U test was used for statistical testing. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The full length (FL), N-terminus double bromodomain (BD, 1-508aa) and C-terminus region containing large segments of low complexity domains (LCDs, 447-798aa) of mouse BRD2 were PCR amplified from the RSV-Flag-mBrd2 vector (Addgene, 86614) and subcloned into a PiggyBac vector containing an N-terminus V5-SNAP tag.

Techniques: CRISPR, Membrane, Labeling, Western Blot, MANN-WHITNEY, Control, Stable Transfection, Expressing, Plasmid Preparation

Journal: Nature genetics

Article Title: BRD2 Compartmentalizes the Accessible Genome

doi: 10.1038/s41588-022-01044-9

Figure Lengend Snippet: (a ) WB test of time and dose-dependent degradation of protein levels of endogenous BRD2-dTAG. 100nM dTAG13 is sufficient to deplete BRD2 as early as 3 hours. We used 100nM dTAG13 treatment for 6 hours throughout the current study. (b-c ) Quantification of single cell fluorescence of endogenously labeled HaloTag-BRD2/3/4-using HaloTag ligand JF 646 before and after dTAG13 treatment for 6 hours (b). RAD21 GFP signal serves as the control (c). (d-f ) Representative images of acute degradation of BRD2 ( d ), BRD3 ( e ) and BRD4 ( f ) after 6 hours of dTAG13 treatment. Endogenous BET proteins were engineered with a HaloTag and labeled with 500nM JF 646 . The RAD21 level was monitored by GFP. Scale bar,5 μm. (g-i) Cell cycle analysis after 6 hours depletion of BRD2, BRD3 or BRD4 by propidium iodide staining. Histograms of the DNA content distribution measured by propidium iodide staining are shown. (j ) WB analysis of protein levels of endogenous BRD3-dTAG or RAD21-AID individually or in combination after 6 hours of dTAG13 or auxin treatment, respectively. (k ) Depletion of BRD3 for 6 hours did not significantly reduce the accessible chromatin clustering after RAD21 depletion. g(r) curves were plotted for indicated conditions. The two-sided Mann-Whitney U test was used for statistical testing. (l) Violin plot of 3D volumes (the number of voxels) of two ATAC-rich segments R2 (left) and R5 (right) before (grey) and after (red) BRD3 depletion, RAD21 depletion or in combination. The number of alleles analyzed is indicated at the bottom. The black bar indicates the median value for each data set and Mann-Whitney U test was performed for the statistical analysis. n.s.,not significant. (m) WB analysis of protein levels of endogenous BRD4-dTAG or RAD21-AID individually or in combination after 6 hours of dTAG13 or auxin treatment, respectively. (n ) Degradation of BRD4 for 6 hours did not significantly reduce but instead had a trend to increase the accessible chromatin clustering after RAD21 depletion. g(r) curves were plotted for indicated conditions. The two-sided Mann-Whitney U test was used for statistical testing. (o) Violin plot of 3D volumes (the number of voxels) of two ATAC-rich segments R2 (left) and R5 (right) before (grey) and after (red) BRD4 depletion, RAD21 depletion or in combination. The number of alleles analyzed is indicated at the bottom. The black bar indicates the median value for each data set and Mann-Whitney U test was performed for the statistical analysis.

Article Snippet: The full length (FL), N-terminus double bromodomain (BD, 1-508aa) and C-terminus region containing large segments of low complexity domains (LCDs, 447-798aa) of mouse BRD2 were PCR amplified from the RSV-Flag-mBrd2 vector (Addgene, 86614) and subcloned into a PiggyBac vector containing an N-terminus V5-SNAP tag.

Techniques: Fluorescence, Labeling, Control, Cell Cycle Assay, Staining, MANN-WHITNEY

Journal: Nature genetics

Article Title: BRD2 Compartmentalizes the Accessible Genome

doi: 10.1038/s41588-022-01044-9

Figure Lengend Snippet: (a ) Snapshots of Micro-C contact maps at different size scales (whole chromosome, 10Mbp, 2Mbp) for Control, Cohesin depletion, BRD2 depletion or in their combination. The number of reads for each condition analyzed are indicated inside the box region in the left panel (M, million reads). Whole chromosome, chromosome 3; 10-Mb region, chromosome1:184M-194M; 2-Mb region, chromosome1:119M-121M. (b-d ) Micro-C analysis of loops ( b ), TADs (c) and compartments ( d ) after Cohesin depletion, BRD2 depletion or in their combination. See more details in the Micro-C analysis in the section. (e) Differential saddle plot analysis of Cohesin depletion, BRD2 depletion or in their combination compared to non-treated control cells. (f) Live cell analysis of cells stably expressing the V5-SNAP tagged full length (FL), N-terminus double bromodomain (BD) and the C-terminus low complexity domain (LCD) of mouse BRD2. Cells were stained with SNAP JF 552 ligand and counterstained with Hoechst 33342. First column indicates the zoomed-in view of cells marked by the while box in the second column. (g) WB analysis of cells from (f) analyzed by ant-V5 antibody and the anti-histone H4 antibody. (h-i ) Micro-C analysis of loops ( h ) and TADs (i) for cells stably expressing FL BRD2 with or without Cohesin depletion. Ectopic expression of FL BRD2 does not restore the loss of Cohesin-dependent chromatin loops or architectural domains. (j ) Micro-C analysis of compartments by the Pearson correlation matrix (chromosome 17) for cells stably expressing FL BRD2 with or without Cohesin depletion. (k) Quantification of the digitalized A-A compartmental interactions (log10 value of observed/expected) after ectopically expressing FL BRD2 with or without RAD21 depletion from Micro-C experiments. The black solid line in each violin plot represents the median value. *** indicates p < 0.001 by the non-parametric Mann-Whitney test.

Article Snippet: The full length (FL), N-terminus double bromodomain (BD, 1-508aa) and C-terminus region containing large segments of low complexity domains (LCDs, 447-798aa) of mouse BRD2 were PCR amplified from the RSV-Flag-mBrd2 vector (Addgene, 86614) and subcloned into a PiggyBac vector containing an N-terminus V5-SNAP tag.

Techniques: Control, Cell Analysis, Stable Transfection, Expressing, Staining, MANN-WHITNEY

Journal: Nature genetics

Article Title: BRD2 Compartmentalizes the Accessible Genome

doi: 10.1038/s41588-022-01044-9

Figure Lengend Snippet: (a) Histogram of eigenvector values from the Pearson’s correlation matrix of single BRD2 or RAD21 depletion and dual RAD21/BRD2 depletion for 6 hours compared to untreated Control from Micro-C experiments. BRD2 depletion increases the switching of B to A compartments. (b ) Browser track view of the eigenvector values for compartmental scores in various perturbation conditions in chromosomes 11 (Chr11). The genomic regions containing B to A switches after BRD2 depletion (green line) are highlighted with black arrows. (c) Live cell single molecule tracking (SMT) of BET proteins by stroboscopic imaging. The jump length fitting of BRD2 dynamics is best described by a three-state model: diffusive, slow (likely transient, non-specific collision) and bound (likely stably bound to cognate sites). The probability distribution function of jump length or single molecule displacement was fit over multiple camera integration time scales. The same three-state model applies to BRD3 or BRD4 (data not shown). (d-e ) The chromatin bound fraction (d) and diffusion coefficient D (e) of BRD2, BRD3 and BRD4 before and after 6 hours of RAD21 depletion were quantified from SMT experiments. The number of cells analyzed are n=17 and n=19 for control and RAD21 depletion for BRD2 SMT, n=17 and n=17 for control and RAD21 depletion for BRD3 SMT, and n=17 and n=19 for control and RAD21 depletion for BRD4 SMT. (f) ChIP-seq analysis of BRD2, BRD3 and BRD4 after 6 hours Cohesin depletion. Shown are the enrichment profile (upper panel) and heatmap (lower panel) of each protein over its binding peaks. (g ) A violin plot showing the log2 fold change of BRD2 CUT&Tag and BRD2/3/4 ChIP-seq intensity at corresponding peaks. Both BRD2 CUT&Tag and BRD2 ChIP-seq show preferential increase at BRD2 ChIP-seq peak regions compared to BRD3/BRD4. (h) Putative model of B to A compartmental switch after removing BRD2. The active A compartment is colored in pink whereas the inactive B compartment in cyan. BRD2 molecules bind to the active A compartment enriched with CTCF and acetylated nucleosomes. BRD2 depletion weakens the boundary resulting in more Cohesin translocation into the neighboring inactive segments and more A/B mixing. (i) Putative model showing the enhanced chromatin binding of BRD2 associated with an increased spatial clustering of ACDs upon Cohesin loss. For simplicity, two small segments containing acetylated nucleosomes are shown. The non-parametric Mann-Whitney U test was used for statistical testing. **, p < 0.01; n.s., not significant.

Article Snippet: The full length (FL), N-terminus double bromodomain (BD, 1-508aa) and C-terminus region containing large segments of low complexity domains (LCDs, 447-798aa) of mouse BRD2 were PCR amplified from the RSV-Flag-mBrd2 vector (Addgene, 86614) and subcloned into a PiggyBac vector containing an N-terminus V5-SNAP tag.

Techniques: Control, Imaging, Stable Transfection, Diffusion-based Assay, ChIP-sequencing, Binding Assay, Translocation Assay, MANN-WHITNEY

Journal: Nature genetics

Article Title: BRD2 Compartmentalizes the Accessible Genome

doi: 10.1038/s41588-022-01044-9

Figure Lengend Snippet: (a) Quantification of compartmental changes after BRD2 and RAD21 depletion individually or in combination. A, active compartments; I, intermediated mixed compartments; B, inactive compartments. The mouse genome is binned into 25480 segments (1Mbp bin size) and the percentage of each fraction is shown. See details of in the section. (b ) Browser track view of the eigenvector values for compartmental score in various perturbation conditions on chromosome 17. The genomic regions containing B to A switches after BRD2 depletion (green line) are highlighted with black arrows. (c ) Endogenous Co-immunoprecipitation between BRD2 and CTCF in mouse ESCs. Benzonase (BZ) treatment did not abrogate the BRD2-CTCF interaction as compared to untreated control (UT). (d ) (Upper panel) Schematic of genome engineering of BET proteins with HaloTag and labeled with Janelia photoactivatable fluorophore PA-JF 549 for single molecule tracking. (Lower panel) Illustration of stroboscopic single molecule imaging of BET protein dynamics in live cells. After activation by a 405 nm pulse, single BET molecules were excited by 561 nm laser pulse (2 ms) to suppress motion blurring and images were captured with ~5ms exposure times. (e ) The probability distribution function of jump length was fit over multiple camera integration time scales by the two-state model estimated by Spot-On program (See ). The two-state diffusive vs bound model does not well account for the dynamics of BRD2 or BRD3/4 (not shown) as compared to the three-state model in . (f-g ) The diffusive fraction ( f ) and its diffusion coefficient ( g ) analysis for BRD2, BRD3 and BRD4 from SMT analysis after Cohesin depletion. Cohesin depletion significantly decreased the diffusive fraction of BRD2 accompanied with an increase of the chromatin bound fraction. The number of cells analyzed are the same as - . (h-i ) The slow bound fraction ( h ) and its diffusion coefficient ( i ) analysis for BRD2, BRD3 and BRD4 from SMT analysis after Cohesin depletion. Cohesin depletion also significantly decreased the diffusion coefficient of slow bound BRD2. The number of cells analyzed are the same as - .

Article Snippet: The full length (FL), N-terminus double bromodomain (BD, 1-508aa) and C-terminus region containing large segments of low complexity domains (LCDs, 447-798aa) of mouse BRD2 were PCR amplified from the RSV-Flag-mBrd2 vector (Addgene, 86614) and subcloned into a PiggyBac vector containing an N-terminus V5-SNAP tag.

Techniques: Immunoprecipitation, Control, Labeling, Imaging, Activation Assay, Diffusion-based Assay

Journal: Nature genetics

Article Title: BRD2 Compartmentalizes the Accessible Genome

doi: 10.1038/s41588-022-01044-9

Figure Lengend Snippet: (a ) Scatter plot of two biological replicates of ChIP-seq signal for BRD2, BRD3 and BRD4. Ranked spearman correlation coefficient is shown above each plot. (b ) Pie diagram of different genomic binding features of BRD2, BRD3 and BRD4 extracted from their respective ChIP-seq peak signal. The percentage of BET proteins binding to different genome features are summarized in the table on the bottom. (c) Enrichment of BET protein ChIP-seq signal at CTCF sites was performed using k-means clustering (n=2, enriched and non-enriched). BRD2 has the highest enrichment profile among BET proteins at CTCF sites at the binding-enriched cluster. (d) (Left panel) Representative genome browser track of Cohesin ChIP-seq (magenta) and BRD2 CUT&Tag (blue) before and after Cohesin depletion (RAD21(−)). Shaded area demonstrates the increased BRD2 signal at Cohesin binding peaks. (Right panel) BRD2 enrichment and heatmap analysis by CUT&Tag (Cell Signaling, 5848S) at Cohesin ChIP-seq peaks before and after Cohesin depletion. (e) Genome wide heatmap analysis of BRD2 CUT&Tag enrichment at Cohesin ChIP-seq peaks before and after Cohesin depletion by using a different antibody (Bethyl laboratories, A302-583A). (f) ChIP-seq analysis of RAD21 and CTCF after 6 hours Cohesin depletion. Shown are the enrichment profile (upper panel) and heatmap (lower panel) of each protein over its binding peaks. (g ) Chromatin fractionation assay of BRD2 and BRD4 in non-treated Control and RAD21 depletion conditions after different salt extraction of ESC nuclei followed by western blot analysis. GAPDH and Histone H4 was used as marker for the cytoplasmic and chromatin bound fraction, respectively. In control cells, BRD2 is preferentially extracted from the 300mM NaCl concentration. After Cohesin depletion, more BRD2 is extracted from the 600mM NaCl concentration. (h ) Summary of the relative BRD2 enrichment in different chromatin fractionations before and after RAD21 depletion from 4 replicates. Each fraction is normalized to histone H4 abundance. * indicates p < 0.05 by non-parametric Mann-Whitney test. Error bar represents standard deviation. The non-parametric Mann-Whitney U test was used for statistical testing. *, p < 0.05.

Article Snippet: The full length (FL), N-terminus double bromodomain (BD, 1-508aa) and C-terminus region containing large segments of low complexity domains (LCDs, 447-798aa) of mouse BRD2 were PCR amplified from the RSV-Flag-mBrd2 vector (Addgene, 86614) and subcloned into a PiggyBac vector containing an N-terminus V5-SNAP tag.

Techniques: ChIP-sequencing, Binding Assay, Genome Wide, Fractionation, Control, Extraction, Western Blot, Marker, Concentration Assay, MANN-WHITNEY, Standard Deviation

Journal: Nature genetics

Article Title: BRD2 Compartmentalizes the Accessible Genome

doi: 10.1038/s41588-022-01044-9

Figure Lengend Snippet: (a) Enrichment profile and heatmap of BRD2/3/4, CTCF, P300 ChIP-seq signal at their respective binding peaks before and after BRD2 depletion. (b) Enrichment profile and heatmap of ChIP-seq signal of BRD2, BRD3, BRD4 and CTCF at their cognate binding peaks after 6 hours of BRD3 depletion. (c) Heatmap of BRD2 binding signal specifically at BRD2/BRD4 co-bound regions before and after acute BRD4 depletion for 6 hours. (d ) Representative genomic tracks (from integrated genomics viewer) of IgG, BRD3 and p300 after BRD4 depletion. BRD4 depletion markedly reduced P300 binding. (e) Validating the specificity of fluorescence signal or antibodies used for immunofluorescence after acute depletion of RAD21 or BRD2/BRD4. The RAD21-mAID-eGFP : BRD2-HA-dTAG : BRD4-Halo-dTAG triple degron mESC line was used in this study. RAD21 was detected by covalently tagged eGFP signal. BRD2 was detected by mouse monoclonal anti-HA antibody and goat-anti-mouse Alexa568 secondary antibody. BRD4 was detected by rabbit BRD4 primary antibody and goat-anti-rabbit Alexa647 secondary antibody. To validate the fluorescence signal specificity, RAD21 and BRD2/BRD4 were depleted by adding auxin (500μM) or dTAG13 (100nM) for 6 hours, respectively. (f) (Upper panel) Representative images of spatial distribution of RAD21, BRD2 and BRD4 signal. (Lower panel) Fluorescent Intensity profile of RAD21(green), BRD2 (cyan) and BRD4 (magenta) relative to DAPI (blue). RAD21 is enriched at both DAPI high and low regions whereas BRD2 and BRD4 are enriched at DAPI low regions. Scale bar, 3μm. (g) Representative single cell view of Cohesin (RAD21, green), BRD2 (cyan) and BRD4 (magenta) in their merged view. The while line indicates the cross-sectional line to analyze the fluorescent intensity profile. The cyan arrow indicates the BRD2 puncta showing little colocalization with BRD4. The magenta arrow indicates the BRD4 puncta poorly colocalized with BRD2. Scale bar, 1μm. (h) Fluorescence intensity profile of Cohesin (RAD21, green), BRD2(cyan) and BRD4 (magenta) along the while line in (g). (i) Whole nucleus 3D voxel-to-voxel correlation summary of RAD21 vs BRD2 and BRD4 vs BRD2 as shown in . The percentage of voxel colocalization of protein pair, the Pearson’s coefficient, and the Manders’ coefficient are computed and summarized.

Article Snippet: The full length (FL), N-terminus double bromodomain (BD, 1-508aa) and C-terminus region containing large segments of low complexity domains (LCDs, 447-798aa) of mouse BRD2 were PCR amplified from the RSV-Flag-mBrd2 vector (Addgene, 86614) and subcloned into a PiggyBac vector containing an N-terminus V5-SNAP tag.

Techniques: ChIP-sequencing, Binding Assay, Fluorescence, Immunofluorescence

Journal: Nature genetics

Article Title: BRD2 Compartmentalizes the Accessible Genome

doi: 10.1038/s41588-022-01044-9

Figure Lengend Snippet: (a) Representative images of BRD2 immunofluorescence before and after acute RAD21 (500μM auxin 6 hours) or BRD4 depletion (100nM dTAG13, 6 hours) in the dual RAD21-mAID-eGFP and BRD4-Halo-dTAG mESC line. BRD2 was detected by anti-BRD2 primary antibody and goat anti-rabbit Alexa568 secondary antibody. (b) Quantification of BRD2 puncta size (left panel) and puncta mean intensity (right panel) in control (n = 1502), RAD21 depletion (n = 2314), BRD4 depletion (n = 2861) conditions, n represent the number of BRD2 puncta analyzed. (c-e ) Micro-C analysis of loops ( c ), TADs ( d) and compartments ( e ) after BRD4 depletion or dual BRD2/BRD4 depletion in the presence or absence of Cohesin. In e , saddle plots of compartmental interactions are shown. (f-g ) Micro-C analysis of active compartmental interactions (A-A interactions) ( f ) and histogram of eigenvalues from the Pearson’s correlation matrix ( g ) for BRD4 depletion alone or dual BRD4/BRD2 depletion. (h-i ) RNA-seq analysis of differentially expressed genes (DEGs) after BRD4 (h) or BRD2 (i) depletion. Volcano plot of significantly (p<0.05) expressed genes after 6 hours depletion of BRD4 or BRD2 are shown. Red dots indicate significant expressed genes with p<0.01. False positive rate adjusted p value in the -log10 form are shown in the y axis. We detected 133 DEGs for BRD2 depletion and 1579 genes for BRD4 depletion. (j) Cumulative distribution function of differentially expressed genes (DEGs) after BRD2 depletion over control relative to compartmental switches. A-A indicates no switch within the A compartment. A-I/B, switches from A to intermediate (I) or B compartment. B-I/A, switches from B to I or A compartment. The overlapping curves suggest no obvious association of DEGs with compartmental changes. (k) Profiling of chromatin accessibility by ATAC-seq before and after acute BRD2 depletion (6 hours) over DEGs (upregulated and downregulated in Fig .(i) ).

Article Snippet: The full length (FL), N-terminus double bromodomain (BD, 1-508aa) and C-terminus region containing large segments of low complexity domains (LCDs, 447-798aa) of mouse BRD2 were PCR amplified from the RSV-Flag-mBrd2 vector (Addgene, 86614) and subcloned into a PiggyBac vector containing an N-terminus V5-SNAP tag.

Techniques: Immunofluorescence, Control, RNA Sequencing

Journal: Nature genetics

Article Title: BRD2 Compartmentalizes the Accessible Genome

doi: 10.1038/s41588-022-01044-9

Figure Lengend Snippet: (a) Representative chromatin conformation snapshots from polymer simulation incorporating both loop extrusion and scaffold protein-mediated interactions. Simulation snapshots represent wild type (WT, incorporating Cohesin, CTCF and scaffold protein), Cohesin depletion (−), scaffold depletion (−) and dual Cohesin and scaffold depletion. See details in the in the section. (b) Polymer simulation integrating both loop extrusion and scaffold protein-mediated dynamic protein-chromatin and protein-protein interactions reproduces enhanced clustering of ACDs upon Cohesin depletion. The pair auto-correlation function g ( r ) for active chromatin beads from all recorded polymer conformations were plotted for WT and Cohesin depletion in which the scaffold protein is present or depleted (−). Removing the scaffold protein decreases the g ( r ) as observed in and . (c) The cumulative distribution function (CDF) value as a function of the 3D loci pair distance from the center of neighboring ATAC-rich segments were extracted in WT (blue curve) and Cohesin depletion (−) plus scaffold protein conditions in which both loop extrusion and scaffold protein dynamic interaction are incorporated in the revised polymer model. (d) Polymer model predictions of the segment volume of ATAC-rich segments under WT and Cohesin depletion (−) plus scaffold protein conditions. The new polymer model recapitulates experimental observations of the volume changes in - . (e) Average contact probability between ATAC-rich regions and ATAC-poor regions for WT, scaffold protein depletion, WT with protein-protein interaction reduced from 2.5k B T to 1.0k B T (non-specific) and WT with protein-protein interaction depleted (0k B T) calculated from simulations. Error bar indicates the range of average contact probability from two ATAC-rich regions from both sides with the ATAC-poor regions in the middle. k B , Boltzmann constant; T, temperature. (f ) Schematic of putative chromatin configurational changes under WT (left), Cohesin depletion (middle) or dual Cohesin/BRD2 depletion (right) conditions. ATAC-rich and ATAC-poor segments are shown in pink and cyan color, respectively. Although Cohesin loss eliminates Cohesin-dependent loops, BRD2 (and others) orchestrates extensive protein-protein and protein-chromatin interactions that lead to the enhanced chromatin interactions between ATAC-rich segments and largely unchanged 3D volume of individual ATAC-rich segments. Further BRD2 depletion mitigates interactions between active chromatin segments and decompacts individual ATAC-rich segments. (g ) Summary model. Cohesin is the master regulator of chromatin loops. BRD4 could associate with NIPBL to enhance chromatin loop formation . Both BRD4 and Cohesin could suppress BRD2 binding to chromatin, which in turn antagonize chromatin compartmentalization in euchromatin.

Article Snippet: The full length (FL), N-terminus double bromodomain (BD, 1-508aa) and C-terminus region containing large segments of low complexity domains (LCDs, 447-798aa) of mouse BRD2 were PCR amplified from the RSV-Flag-mBrd2 vector (Addgene, 86614) and subcloned into a PiggyBac vector containing an N-terminus V5-SNAP tag.

Techniques: Polymer, Protein-Protein interactions, Binding Assay

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, RNA-Seq–derived gene expression levels from TCGA were analyzed by UALCAN portal. Box plot analysis shows relative expression of AKAP95 in 28 types of cancer (red box) versus normal (blue box) samples, unless indicated for different cancer stages or tumor grades for certain cancer types. Cancer type in red font has significantly higher AKAP95 expression in cancer than in normal (or in later stage than in earlier stage). Cancer type in blue font has significantly lower AKAP95 expression in cancer than in normal samples (or in later stage than in earlier stage). Numbers on the left of the plot stand for the number of samples. Data are median (line), 25–75th percentiles (box) and minimum-maximum values recorded (whiskers). b, Assays for cell proliferation by BrdU incorporation (top) and apoptosis by Annexin V staining (bottom) for control and AKAP95-KD MDA-MB-231 cells. Images of flow cytometry results are shown (left), and percentages of cells positive for BrdU or Annexin V are shown as mean ± SD from n = 3 independent KD assays. c, MDA-MB-231 cells were infected to express scramble (control) or AKAP95 shRNA #1 (KD) and indicated constructs. Top, immunoblotting of total cell lysates. Middle, images of these cells seeded at high (5 ×10 4 cells/well in 6-well plate, top) and low (400 cells/well in 24-well plate, bottom) densities and stained with crystal violet. Bottom, cell numbers (seeded at high densities) as mean from 2 independent experiments. d, MCF7 cells were infected to express scramble control shRNA or two AKAP95 shRNAs. Bottom, images of indicated cell colonies stained with crystal violet. Top, cell numbers were quantified and presented in the bar graph as mean from 2 independent experiments. e, Top 10 gene sets enriched in genes down- (top, n = 951 genes) and up- (bottom, n = 294 genes) regulated by AKAP95 KD in MDA-MB-231 cells. NES, normalized enrichment score. P values by two-sided Student’s t -test for a-d and modified Fisher’s exact test for e. Uncropped blots and statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: RNA Sequencing Assay, Derivative Assay, Expressing, BrdU Incorporation Assay, Staining, Flow Cytometry, Infection, shRNA, Construct, Western Blot, Modification

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, Overexpression of AKAP95 in breast cancer tissues of 82 TNBC patient samples. From cBioPortal. Top, each box is a patient sample. Bottom, disease-free survival curves of patients with or without AKAP95 alterations. n = 17 and 65 patient samples for AKAP95 altered and not altered, respectively. b, Growth assay for MDA-MB-231 cells expressing control or two AKAP95 shRNAs. Left, immunoblotting of total cell lysates and images of cell colonies stained with crystal violet. Right, numbers of cells in growth assays as mean ± SD from n = 3 independent experiments. c, Tumors from xenograft of control or AKAP95-KD MDA-MB-231 cells in immune-deficient mice. Tumor volumes at the indicated days post transplantation are plotted as mean ± SD (n = 9 mice). d,e, RNA-seq analysis in MDA-MB-231 cells expressing control or AKAP95 shRNA #1 and the indicated vector or AKAP95-expressing construct. One representative analysis from 2 repeats. d, Heatmap showing relative expression levels of genes down- or up-regulated in the indicated cells. It includes 951 and 294 genes down- and up-regulated in KD compared to control cells, respectively. Also see . e, GSEA for gene expression profiles of control and AKAP95-KD cells. Plots above and below the broken line show gene sets significantly enriched in up- and down-regulated genes by AKAP95 KD, respectively. f, Heatmap showing relative alternative splicing and clustered by changes in percent-spliced-in (PSI) values in the indicated cells. It includes 807 and 1275 alternative splicing events with decreased or increased PSI in KD cells, respectively. Also see . g, Gene ontology analysis for the indicated clusters from the heatmap in f. Blue (n = 807 genes) and red (n = 1275 genes) show functions significantly enriched in genes with PSI increase or decrease by AKAP95 KD, respectively. P values by log-rank test for a, Student’s t -test for b and d, and modified Fisher’s exact test for g. All two-sided. Uncropped blots and statistical source data are provided in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Over Expression, Growth Assay, Expressing, Western Blot, Staining, Transplantation Assay, RNA Sequencing Assay, shRNA, Plasmid Preparation, Construct, Modification

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, CCNA2 expression in MDA-MB-231 cells upon AKAP95 KD. Left, relative mRNA levels of indicated cyclins were determined by RT-qPCR and normalized to GAPDH , and presented as mean ± SD from n = 3 biological repeats. Right, immunoblotting for Cyclin A1/A2. b, Co-overexpression of AKAP95 and CCNA2 in breast cancer tissues of TNBC patients. Left, each box represents a patient. Right, correlation of their mRNA levels in the TNBC patients with indicated Pearson correlation coefficient. From cBioPortal. n = 82 patient samples. c,f, RNA immunoprecipitation-sequencing (RIP-seq) profiles for CCNA2 (c) and SMAD6 (f) based on our previous work . Blue, anti-FLAG RIP-seq in control or 293 cells expressing the FLAG-HA-tagged AKAP95 WT or mutants. Red, anti-AKAP95 RIP-seq in control or AKAP95-KD 293 cells. Black, profiles of total input RNAs. All profiles have the same Y-axis scale. Arrows indicate AKAP95-binding sites at intron 1. One representative RIP-seq analysis from 2 repeats. d, Total RNAs were used for RT-PCR, in the absence (- RT) or presence (+ RT) of reverse transcriptase, for CCNA2 intron 1 in MDA-MB-231 cells with indicated siRNAs. Top, PCR products on agarose gel. Asterisk, an unknown amplification product. Repeated 3 times. Bottom, relative ratios of the signal for the intron 1- retaining transcript over the intron 1-spliced transcript, as mean ± SD from n = 3 independent experiments. e, Assay for CCNA2 mRNA stability. Control (Scr) and AKAP95-KD MDA-MB-231 cells were treated starting from 0 min with Actinomycin D (+A, to block RNA synthesis) and cycloheximide (+C, to block NMD) or not as indicated. Total RNA at indicated times were used for RT-PCR and normalized to ACTB , as mean ± SD from n = 3 biological repeats. g, mRNA-seq profiles for SMAD6 in MDA-MB-231 cells expressing control or AKAP95 shRNA #1 (KD) and vector or AKAP95-expressing construct. Asterisk, a stop codon. P values by two-sided Student’s t -test for a and e and one-way ANOVA followed by Tukey’s post hoc test for d. Uncropped blots and statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Over Expression, Immunoprecipitation, Sequencing, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Blocking Assay, shRNA, Plasmid Preparation, Construct

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a,b, MDA-MB-231 cells virally expressing control or indicated shRNAs ( a ) or shRNA combined with indicated constructs ( b ) were subject to immunoblotting of total cell lysates (top) and colony formation assay. Middle, colony numbers as mean ± SD from n = 3 ( a ) or mean from 2 ( b ) independent experiments. Bottom, images of cells stained with crystal violet. c, Top, mRNA-seq profiles for CCNA2 in control or AKAP95 KD 293 cells . The numbers of exon junction reads are indicated. The red asterisk at the gene diagram indicates a stop codon 57 bp downstream of exon 1 in the intron. The number of reads for the junction of exons 1 and 2, and for the average neighboring exons, and their ratios are in the tables below for indicated cells. d, Total RNAs were used for RT-PCR for intron 1 region in control and AKAP95-KD MDA-MB-231 cells treated with or without cycloheximide for 6 hours. Repeated 3 times. e, Relative mRNA levels of UPF in UPF1-KD samples and BTZ in BTZ-KD samples, respectively, each relative to the control samples, as determined by RT-qPCR and normalized to GAPDH . Mean is from 2 independent experiments. f, Relative expression level of TGF-β pathway genes based on RNA-seq reads from control and AKAP95-KD MDA-MB-231 cells expressing vector or AKAP95. Venn diagram shows numbers of TGF-β pathway genes (from GSEA) downregulated by AKAP95 KD and upregulated by rescue with AKAP95 expression, and the relative expression of the 16 overlapped genes in both categories are plotted. One representative analysis from 2 repeats. g, RIP-seq profiles showing AKAP95 binding to RPUSD3 and PPM1K pre-mRNAs. Track information is the same as in . Red circles indicate the alternatively included exons (corresponding to the middle exon in the gene diagrams in ( h ), and red boxes show AKAP95 binding at the introns flanking these exons. h, Sashimi plots showing that the alternative splicing of RPUSD3 and PPM1K pre-mRNAs was affected by AKAP95 KD and rescued by restored expression of AKAP95. The numbers of exon junction reads and PSI are indicated. P values by two-sided Student’s t -test for a and b. Uncropped blots and statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Expressing, shRNA, Construct, Western Blot, Colony Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, RNA Sequencing Assay, Plasmid Preparation, Binding Assay

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, Schematic of the “Knockout-first” Akap95 allele (null without further recombination). b, Body weights of mice with indicated Akap95 genotypes. Not significant (n.s.) between any two groups at any time for male, or after week 26 for female. *between Het and KO before week 26 for female. c, Peripheral blood profiles of 8-week mice of indicated genotype. n in b, c refers to the number of mice analyzed. d, MEFs were derived from mouse embryos. Images of six embryos from the same litter were shown at the top, followed with genotyping results, Ponceau S staining, and immunoblotting of MEFs. e, Top, heatmap showing relative expression levels of genes and clustered by changes in un-transduced KO MEFs from 2 embryos each. It includes 203 and 20 genes down- or up-regulated in KO, respectively. Bottom, heatmap showing relative alternative splicing of genes clustered by PSI changes. It includes 285 and 332 alternative splicing events with decreased or increased PSI in KO, respectively. Also see and . f, Top 10 gene sets enriched in genes down- (left, n = 265 genes) and up- (right, n = 742 genes) regulated in the MYC-transduced KO versus Het MEFs. g, Rescue of the gene expression profile by introduction of human AKAP95 into the MYC-transduced KO MEFs as shown by immunoblotting and heatmap for relative expression of down- or up-regulated genes in the indicated cells. Also see . Repeated 2 times. h, Relative expression of Akap95 and Akap8l in un-transduced (-MYC) and MYC-transduced (+MYC) MEFs from n = 2 Het and two KO embryos, as determined by normalized RNA-seq reads. i, Heatmap showing relative alternative splicing of genes clustered by PSI changes in MYC-transduced MEFs from 2 embryos each. It includes 216 and 252 alternative splicing events with decreased or increased PSI in KO versus Het MEFs, respectively. Also see . j, Gene ontology analysis for the indicated gene clusters from the heatmap in i . Blue (n = 216 genes) and red (n = 252 genes) show functions significantly enriched in genes with PSI increase or decrease by KO, respectively. k, Sashimi plots showing alternative splicing changes for each gene cluster from the heatmap using two examples, Asb7 for cluster 1, and Xpo4 for cluster 2. ns, not significant, *P < 0.05, by two-sided Student’s t -test for b, one-way ANOVA for c and modified Fisher’s exact test for f, j. Uncropped blots and statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Knock-Out, Derivative Assay, Staining, Western Blot, Expressing, RNA Sequencing Assay, Modification

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, Growth of MEFs from Akap95 +/- (Het) and Akap95 −/− (KO) embryos (n = 6 embryos each). b, Relative HRAS and MYC mRNA levels by RT-qPCR and normalized to Actb , as mean from HRAS G12V and MYC transduced MEFs (2 embryos each). c, HRAS-MYC-transduced MEFs in colony formation assay. Colony numbers as mean ± SD from n = 6 experiments using MEFs of 2 embryos each. d, Six mice received HRAS-MYC-transduced Het and KO MEFs on each flank. Tumor weights (week 4) are plotted. Each dot represents a tumor. e-i, MYC-transduced MEFs from 3 KO and 3 Akap95-expressing (1 WT, 2 Het) embryos. e, Right, images of cells before and after MYC transduction. Images of SA-beta-galactosidase activity assay are at bottom. Relative MYC mRNA levels after transduction were determined by RT-qPCR and normalized to Actb (left top). Percentage of SA-beta-gal-positive cells are plotted (left bottom). Both as mean ± SD from MEFs (n = 3 embryos each). f, Heatmap showing relative expression of genes and clustered by changes in KO MEFs (2 embryos each separately analyzed), with 265 and 742 genes down- or up-regulated in KO, respectively. Also see . g, Gene ontology analysis for the indicated gene clusters from the heatmap in f. Blue (n = 265 genes) and red (n = 742 genes) show functions significantly enriched in down- and up-regulated genes, respectively. h, GSEA plots above and below dashed line show gene sets significantly enriched in genes down- and up-regulated in the MYC-transduced KO compared to Het MEFs, respectively. i, Relative Akap95 and Ccna2 mRNA levels before and after MYC transduction by RT-qPCR and normalized to Actb , as mean ± SD from MEFs from n = 3 embryos each. j, Diagram summarizing regulation of tumorigenesis by AKAP95 through gene expression control. P values by two-sided Student’s t -test for all except one-way ANOVA followed by Tukey’s post hoc test for i, and modified Fisher’s exact test for g. Statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Quantitative RT-PCR, Colony Assay, Expressing, Transduction, Activity Assay, Modification

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, Immunoblotting for AKAP95 in HeLa cell nuclear extract and AKAP95 immunoprecipitation from the extract. Samples were boiled in the presence of DTT and resolved by SDS-PAGE. b, Disorder plot of human AKAP95. c, Turbidity by pictures and OD600 of MBP (none) and MBP fused to AKAP95 truncations at indicated concentrations all in 30 mM NaCl before and after TEV protease treatment for indicated time. OD600 is plotted as mean ± SD from n = 3 biological repeats. d, DIC (top) and fluorescence microscopy (bottom) images for 20 μM MBP-AKAP95 (101–210) and spiked with Oregon-green-labeled same protein (molar ratio 10:1) after TEV protease treatment for 30 min. Changes in NaCl concentration is indicated. Images were taken 5 min after salt adjustment. e, Phase contrast images of 50 μM MBP-AKAP95 (101–210) in 30 mM NaCl in the absence and presence of 10% of PEG6000 after TEV protease treatment for 30 min. f, Fusion of two droplets formed by 50 μM MBP-AKAP95 (101–210) in 30 mM NaCl and 10% of PEG6000 after TEV protease treatment for 30 min. Also see . g, DIC and fluorescence microscopy images of 6.25 μM MBP, MBP fused to Δ(101–210) or full-length AKAP95 in 150 mM NaCl, spiked with Oregon-green-labeled AKAP95 (101–210) at a molar ratio of 150:1 after TEV protease treatment for 30 min. Note that the lack of any condensates in the DIC images showed the inability of Δ(101–210) in condensation. Experiments in a, d, e-g were repeated 4 times. Scale bar, 5 μm for all. Uncropped blots and statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Western Blot, Immunoprecipitation, SDS Page, Fluorescence, Microscopy, Labeling, Concentration Assay

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, 293T cells were transfected with either empty vector (vec), or indicated AKAP95 construct with FLAG-HA-tag. Following α-Flag IP, the pulldown proteins were boiled and resolved by SDS-PAGE and detected by immunoblotting with α-HA. Blue and red asterisks indicate monomer and dimer, respectively. b, Identification of 1–100 as a probable prion subsequence on AKAP95. By the PLAAC program, using homo sapiens as background and core length of 30. c, Purified MBP and MBP fused to AKAP95 truncations as indicated or full-length AKAP95 (1–692) were resolved on SDS-PAGE and stained with Coomassie blue. d, MBP fused to AKAP95 truncations as indicated or full-length AKAP95 were resolved on SDS-PAGE and stained with Coomassie blue following treatment with TEV protease. Note that the cleaved MBP serves as a better indicator for cleavage efficiency as staining signal various for protein fragments of different sequences and sizes. e, Another event of fusion of two droplets formed by 50 μM MBP-AKAP95 (101–210) in 30 mM NaCl and 10% of PEG6000 after treatment with TEV protease for 30 min. Scale bar, 5 μm. Also see . f, Quantification of nuclear AKAP95 concentration by anti-AKAP95 Western blot. Total lysates from indicated number of MDA-MB-231 (M231) and flp-TREx 293 cells (f293, un-induced and dox-induced for FH-AKAP95 expression) were loaded, along with indicated ng of purified MBP-AKAP95. AKAP95 signal of un-induced f293 is similar to that of 25 ng of MBP-AKAP95. All experiments were repeated 2 times. Uncropped blots are provided as in source data .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Transfection, Plasmid Preparation, Construct, SDS Page, Western Blot, Purification, Staining, Concentration Assay, Expressing

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, Immunostaining of endogenous AKAP95 (red) and DNA (DAPI, blue) in indicated cancer cell lines and primary MEFs from WT and Akap95 KO embryos. b, Confocal microscopy images of AKAP95 WT or ZF C-S fused to GFP in nuclei following transfection into HeLa cells. c, Fluorescence microscopy images of HeLa cells transiently expressing AKAP95 WT or Δ(101–210 fused to GFP. d, HeLa cells were transfected with AKAP95-GFP, and two nuclei were imaged at different time points. Time 0 was 24 hr after transfection. Note the growth and merge of the foci, especially those in the red circle. e, Rapid fusion of AKAP95 (ZF C-S )-GFP foci in a HeLa cell nucleus. The white oval and arrow show two different fusion events. These images are from . All experiments were Repeated 4 times. Scale bar, 5 μm for all.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Immunostaining, Confocal Microscopy, Transfection, Fluorescence, Microscopy, Expressing

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: Fluorescence microscopy images of HeLa cells transiently expressing AKAP95-GFP. Nucleus DNA was stained by DAPI, and specific proteins were stained with antibodies for SRSF2 ( a ), Pol II ( b ), and Pol II-S2P ( c ). The assays were repeated 10 times for each staining and show similar trend. Right, quantification of the signal intensity of indicated molecules across the dotted lines shown in the images. Quantification by Image J. Scale bar, 5 μm for all. Statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Fluorescence, Microscopy, Expressing, Staining

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, Alignment of human and mouse AKAP95 (101–210). Middle row shows identical residues (letter) and conservative mutations (“+”). Tyr, red; Phe, blue and tall. Box, Tyr and Phe swapping. b, MBP alone (none) or MBP-AKAP95 (101–210) WT or mutants all at 50 μM and in 30 mM NaCl after TEV protease treatment for 30 min. Turbidity of each reaction was shown, and by OD600 as mean ± SD from n = 3 (for YA, YS) or 4 (the rest) independent assays. Samples taken after mixing and from supernatant after centrifugation were resolved by SDS-PAGE followed by coomassie blue staining. c, DIC and fluorescence microscopy images for 10 μM Oregon-green-labeled MBP-AKAP95 (101–210) WT and mutants in 30 mM NaCl after TEV protease treatment for 30 min. Plots from left to right show relative protein amount in droplet, number of droplets in a field, and ratio of protein concentration inside droplets over sum of inside and outside droplets, respectively, as mean ± SD from n = 24 randomly picked droplets, except for number of droplets from n = 3 randomly picked fields, in one representative assay from 5 repeats. NA, not applicable. d, Fluorescence microscopy images of HeLa cells (top) and Flp-In T-Rex 293 cell lines expressing (bottom) GFP fusions with full-length AKAP95 WT or mutants. Repeated 4 times. e,h, HEK293 cells co-transfected with indicated siRNAs and plasmids were subject to splice reporter assay (top) and immunoblotting with α-AKAP95 (bottom). Δ = Δ(101–210). Mean ± SD from n = 8 [except 5 for Δ(101–210) and 13 for YF and 2 nd WT] independent transfections are plotted in e and 7 independent transfections in h. f, Schematic of AKAP95 chimeras. g, Fluorescence microscopy images of 293T cells transfected with indicated AKAP95 chimeras fused to GFP. Repeated 3 times. P values by two-sided Student’s t -test for b and one-way ANOVA followed by Tukey’s post hoc test for e and h. Scale bar, 5 μm for all. Uncropped blots and statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Centrifugation, SDS Page, Staining, Fluorescence, Microscopy, Labeling, Protein Concentration, Expressing, Transfection, Reporter Assay, Western Blot

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, Amino acid enrichment for AKAP95 (101–210). By Composition Profiler, using SwissProt 51 Dataset as background. b, Purified MBP fused to AKAP95 (101–210) WT and mutants were resolved on SDS-PAGE and stained with Coomassie blue. Repeated 2 times. c, Disorder plot of AKAP95 WT or mutants with indicated mutations in 101–210. d, MBP-AKAP95 (101–210) WT, YS, and YF, all at 35 μM and in 30 mM NaCl, were treated with TEV protease for 2 hrs in 3 independent assays, and subjected to centrifugation. The supernatant and pellets (resuspended in the same volume as the supernatant) were resolved by SDS-PAGE followed with coomassie blue staining. MBP signal in the pellet reflects residual supernatant fraction, and its percentage [MBP pellet/(supernatant + pellet)] was subtracted from the (101–210) pellet percentage. Such normalized (101–210) pellet percentages are plotted as Partition Percentage as mean ± SD of n = 3 independent experiments. It is most likely that all supernatants may also have substantial portion of condensates. Moreover, the size cutoff of condensates is also arbitrary, as protein assemblies may take a continuum of size distribution . e, Immunoblotting by α-AKAP95 (top) or GAPDH (bottom) of total lysates from Flp-In T-Rex 293 cell lines induced to express full-length AKAP95 WT or indicated mutants fused to GFP. Repeated 3 times. f,g, Indicated full-length AKAP95 WT or mutants fused to GFP were induced by various concentrations of doxycycline in Flp-In T-Rex 293 cell lines. Immunoblotting of total cell lysates with indicated antibodies ( f ). The doxycycline concentrations in red font activated the transgene at the near endogenous level, and were selected for treating cells and fluorescence microscopy assays of fixed cells in ( g ). Scale bar, 5 μm. Repeated 2 times. h, 293T cells transiently expressing indicated constructs with FLAG-HA-tag were used for α-FLAG immunoprecipitation and immunoblotting with indicated antibodies and Ponceau S staining. Repeated 2 times. i, Immunoblotting of 293T cells transfected with empty vector or indicated FLAG-HA-tagged AKAP95 chimeras fused to GFP. Bottom, by anti-GAPDH. Top, by anti-AKAP95 (Bethyl Laboratories, A301–062A, recognizes an epitope in a region between residue 575 and 625 of human AKAP95). Repeated 2 times. P values by two-sided Student’s t -test for d. Uncropped blots and statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Purification, SDS Page, Staining, Centrifugation, Western Blot, Fluorescence, Microscopy, Expressing, Construct, Immunoprecipitation, Transfection, Plasmid Preparation

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, OD450 at different time after TEV protease treatment of 50 μM MBP-AKAP95 (101–210) WT, YS, and YF in 150 mM NaCl, as mean ± SD of readings after subtracting that of MBP at each time (constant at 0.07–0.08) from n = 3 independent assays. Dashed lines show half of the maximum turbidity and time (τ 1/2 ) to reach it. b, Ratio of protein concentration inside the droplets over sum of inside and outside for (101–210) WT and YF at increasing protein concentrations and in 30 mM NaCl, as mean ± SD from n = 6 randomly picked droplets each, in one representative assay from 3 repeats based on . c, Confocal microscopy images of GFP-AKAP95 (101–210) WT and YF at increasing protein concentrations, all in 150 mM NaCl and 10% of PEG6000 after TEV protease treatment for 20 min. Repeated 2 times with similar results. d, DIC and fluorescence microscopy images for 20 μM MBP-AKAP95 (101–210) YF spiked with Oregon-green-labeled same protein (molar ratio 10:1), after TEV protease treatment for 30 min. Changes in NaCl concentration is indicated. Images were taken 5 min after salt adjustment. Repeated 2 times with similar results. e, Different extent of droplet fusion (arrows) by AKAP95 (101–210) WT and YF, both at 50 μM and in 30 mM NaCl and 10% of PEG6000 after TEV protease treatment for 30 min. A similar trend was observed in 5 fusion events (or attempted fusion for YF) for each. Also see – . f, Equation for Line Raster Scan Image Correlation fitting autocorrelation G(Ψ), which depends on G(0)=γ/N (γ: beam profile, N = number of mobile particles), diffusion coefficient D (μm 2 /s), line scan time t l , pixel size Ψ, radial beam waist w 0 and axial beam waist w z . The radial waist w 0 (0.218 μm) was calibrated with sub-diffraction beads (0.1 μm) diluted solution as reported before . The axial waist w z was considered equal to 3*w 0 . g, Fluorescence confocal microscope image of HeLa cell expressing full-length AKAP95 WT or YF fused to GFP. Arrow indicates the scanned region for in vivo Line RICS. h, Fluorescence confocal microscope image of GFP-AKAP95 (101–210) WT and YF in 150 mM NaCl and 10% of PEG6000 after TEV protease treatment for 20 min, for in vitro Line RICS experiments. i, Line Fluorescence carpet formed by ~10 4 lines (each line is composed by 128 pixels, 50 nm/pixel) acquired with 0.101 s line scan time and 32.8 μs/pixel. Line RICS autocorrelation curves were computed on 64 sections of 128 lines followed by averaging all the curves. The sectioning of the line carpet permitted to avoid the effect of the movement of the condensates on the measurement of the autocorrelation curves. (λex=488 nm). j, Line RICS autocorrelation curves for experimental data and fitted. k, Residuals of the fitting. Experiments in g-i were repeated 3 times with similar results. Scale bar, 2 μm for c and h, 5 μm for d, e, and g.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Protein Concentration, Confocal Microscopy, Fluorescence, Microscopy, Labeling, Concentration Assay, Diffusion-based Assay, Expressing, In Vivo, In Vitro

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, Fluorescence microscopy images of Oregon-green-labeled AKAP95 (101–210) WT and YF at indicated protein and NaCl concentrations after MBP cleavage for 30 min. Repeated 3 times. b, Fluorescence microscopy images of 50 μM AKAP95 (101–210) WT and YF in 30 mM NaCl both spiked with Oregon-green-labeled (101–210) WT (molar ratio 150:1) after MBP cleavage for 30’ and imaged immediately (30’) or after incubation for 60 (90’) or 120 (150’) more minutes. Repeated 3 times. c, FRAP of 10 μM GFP-AKAP95 (101–210) WT and YF after 30 min of MBP cleavage in 150 mM NaCl. FRAP was performed immediately (30’) or after incubation for 60 min more (90’). Left, fluorescence microscopy images of droplets at indicated times. Middle, FRAP recovery curves as mean ± SD. Right, mean ± SD of recovery at the final time. n = 7 independent measurements. d, FRAP of Full-length AKAP95 WT and YF fused to GFP in HeLa cell nuclei. Left, fluorescence microscopy images of foci. The photobleached focus was boxed and amplified for indicated time points. Middle, FRAP recovery curves as mean ± SD. Right, mean ± SD of recovery (relative to minimal level) at the final time. n = 7 independent measurements. e,f, Diffusion of full length AKAP95 WT and YF fused to GFP in HeLa cell nuclei, showing Line RICS normalized autocorrelation curves G(Ψ) as function of Spatial Lag (Ψ) ( e ) and diffusion coefficients ( f ), as mean ± SD (n = 20 or 22 independent measurements for WT, YF, respectively). g,h, Diffusion coefficients of purified GFP-AKAP95 (101–210) WT and YF, showing Line RICS normalized autocorrelation curves G(Ψ) ( g ) and diffusion coefficients ( h ), as mean ± SD (n = 22 or 13 independent measurements for WT, YF, respectively). Scale bars, 10 (a, b), 2 (c), 5 (d) μm. P values by two-sided Student’s t -test (c, d) or Mann-Whitney U test (f, h). For box-and-whisker plots, data are median (line), 25–75th percentiles (box) and minimum-maximum values recorded (whiskers). Statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Fluorescence, Microscopy, Labeling, Incubation, Amplification, Diffusion-based Assay, Purification, MANN-WHITNEY, Whisker Assay

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a-f, MDA-MB-231 transduced with control or AKAP95 shRNA #1 (KD) and vector or FLAG-HA-tagged full-length AKAP95 WT or mutants. n = 3 biological repeats for a-f, except n = 4 biological repeats for e. a, Immunoblotting of total cell lysates. b, Colony formation assays. Left, colony numbers as mean ± SD. Right, images of cells stained with crystal violet. c, Growth of cultured cells, as mean ± SD. d, Relative SMAD6 mRNA level were determined by RT-qPCR and normalized to GAPDH , as mean ± SD. e,f, RT-PCR for ratios for intron 1-retained over -spliced CCNA2 (e) and exon-included over -skipped RPUSD3 transcripts, as mean ± SD. g-k, MYC-transduced Akap95 KO MEFs transduced with vector or HA-tagged full-length AKAP95 WT or mutants. g, Immunoblotting of total cell lysates. Repeated 3 times. h, Left, percentage of SA-beta-gal-positive cells as mean ± SD (n = 3 different images of MEFs from two embryos). Right, images from KO MEF 1. i, Relative mRNA levels of indicated genes with related functions at bottom by RT-qPCR and normalized to Gapdh , as mean ± SD (n = 3 independent experiments). * or ** between vec and WT, WT and YS, WT and YF, except for Plk1, for which * only between vec and WT, WT and YF. *P<0.05, **P<0.01. j, Heatmap showing relative alternative splicing with PSI changes in MYC-transduced KO MEFs expressing indicated constructs (2 embryos each). Also see . k, Sashimi plot showing Aamdc alternative splicing that was rescued by introduction of AKAP95 WT, but not but the mutant, and RT-PCR for the inclusion of the alternative exon as mean ± SD (n = 4 biological replicates pooled from 2 embryos each). l, Diagram showing impact of material properties of AKAP95 WT and mutants on gene regulation and tumorigenesis. P values by two-sided Student’s t -test for c and one-way ANOVA followed by Tukey’s post hoc test for all other analyses. Uncropped blots and statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Transduction, shRNA, Plasmid Preparation, Western Blot, Staining, Cell Culture, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Expressing, Construct, Mutagenesis

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a,b, MDA-MB-231 cells were virally infected to stably express scramble (control) or AKAP95 shRNA #1 (KD) and the indicated constructs including empty vector (vec) and FLAG-HA-tagged full-length AKAP95 WT or mutants. a, Relative CCNA2 mRNA level as determined by RT-qPCR and normalized to GAPDH , and plotted for each of the 2 biological repeats individually. b, RT-PCR for ratios for exon-included over -skipped PPM1K transcript, as mean ± SD from n = 3 biological repeats. c-f, MYC-transduced Akap95 KO MEFs were transduced with vector or constructs expressing HA-tagged full-length AKAP95 WT or mutants. c, Heatmap showing relative expression levels of genes changed in MYC-transduced KO MEFs (from 2 embryos each) stably expressing indicated rescue constructs. Also see . d, Relative mRNA levels of indicated SASP genes as determined by RNA-seq reads from 2 biological repeats (KO1 and KO2). e,f, Sashimi plots showing example genes for which the alternative exon inclusion was promoted ( e ) or suppressed ( f ) by introduction of AKAP95 WT, but not as effectively by YS or YF, and RT-PCR for the inclusion of the alternative exon, as mean from 2 embryos each. g, A model for how AKAP95 condensates may regulate gene expression for tumorigenesis. P values by one-way ANOVA followed by Tukey’s post hoc test. Uncropped blots and statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Infection, Stable Transfection, shRNA, Construct, Plasmid Preparation, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Transduction, Expressing, RNA Sequencing Assay

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, RNA-Seq–derived gene expression levels from TCGA were analyzed by UALCAN portal. Box plot analysis shows relative expression of AKAP95 in 28 types of cancer (red box) versus normal (blue box) samples, unless indicated for different cancer stages or tumor grades for certain cancer types. Cancer type in red font has significantly higher AKAP95 expression in cancer than in normal (or in later stage than in earlier stage). Cancer type in blue font has significantly lower AKAP95 expression in cancer than in normal samples (or in later stage than in earlier stage). Numbers on the left of the plot stand for the number of samples. Data are median (line), 25–75th percentiles (box) and minimum-maximum values recorded (whiskers). b, Assays for cell proliferation by BrdU incorporation (top) and apoptosis by Annexin V staining (bottom) for control and AKAP95-KD MDA-MB-231 cells. Images of flow cytometry results are shown (left), and percentages of cells positive for BrdU or Annexin V are shown as mean ± SD from n = 3 independent KD assays. c, MDA-MB-231 cells were infected to express scramble (control) or AKAP95 shRNA #1 (KD) and indicated constructs. Top, immunoblotting of total cell lysates. Middle, images of these cells seeded at high (5 ×10 4 cells/well in 6-well plate, top) and low (400 cells/well in 24-well plate, bottom) densities and stained with crystal violet. Bottom, cell numbers (seeded at high densities) as mean from 2 independent experiments. d, MCF7 cells were infected to express scramble control shRNA or two AKAP95 shRNAs. Bottom, images of indicated cell colonies stained with crystal violet. Top, cell numbers were quantified and presented in the bar graph as mean from 2 independent experiments. e, Top 10 gene sets enriched in genes down- (top, n = 951 genes) and up- (bottom, n = 294 genes) regulated by AKAP95 KD in MDA-MB-231 cells. NES, normalized enrichment score. P values by two-sided Student’s t -test for a-d and modified Fisher’s exact test for e. Uncropped blots and statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: RNA Sequencing Assay, Derivative Assay, Expressing, BrdU Incorporation Assay, Staining, Flow Cytometry, Infection, shRNA, Construct, Western Blot, Modification

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, Overexpression of AKAP95 in breast cancer tissues of 82 TNBC patient samples. From cBioPortal. Top, each box is a patient sample. Bottom, disease-free survival curves of patients with or without AKAP95 alterations. n = 17 and 65 patient samples for AKAP95 altered and not altered, respectively. b, Growth assay for MDA-MB-231 cells expressing control or two AKAP95 shRNAs. Left, immunoblotting of total cell lysates and images of cell colonies stained with crystal violet. Right, numbers of cells in growth assays as mean ± SD from n = 3 independent experiments. c, Tumors from xenograft of control or AKAP95-KD MDA-MB-231 cells in immune-deficient mice. Tumor volumes at the indicated days post transplantation are plotted as mean ± SD (n = 9 mice). d,e, RNA-seq analysis in MDA-MB-231 cells expressing control or AKAP95 shRNA #1 and the indicated vector or AKAP95-expressing construct. One representative analysis from 2 repeats. d, Heatmap showing relative expression levels of genes down- or up-regulated in the indicated cells. It includes 951 and 294 genes down- and up-regulated in KD compared to control cells, respectively. Also see . e, GSEA for gene expression profiles of control and AKAP95-KD cells. Plots above and below the broken line show gene sets significantly enriched in up- and down-regulated genes by AKAP95 KD, respectively. f, Heatmap showing relative alternative splicing and clustered by changes in percent-spliced-in (PSI) values in the indicated cells. It includes 807 and 1275 alternative splicing events with decreased or increased PSI in KD cells, respectively. Also see . g, Gene ontology analysis for the indicated clusters from the heatmap in f. Blue (n = 807 genes) and red (n = 1275 genes) show functions significantly enriched in genes with PSI increase or decrease by AKAP95 KD, respectively. P values by log-rank test for a, Student’s t -test for b and d, and modified Fisher’s exact test for g. All two-sided. Uncropped blots and statistical source data are provided in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Over Expression, Growth Assay, Expressing, Western Blot, Staining, Transplantation Assay, RNA Sequencing Assay, shRNA, Plasmid Preparation, Construct, Modification

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, CCNA2 expression in MDA-MB-231 cells upon AKAP95 KD. Left, relative mRNA levels of indicated cyclins were determined by RT-qPCR and normalized to GAPDH , and presented as mean ± SD from n = 3 biological repeats. Right, immunoblotting for Cyclin A1/A2. b, Co-overexpression of AKAP95 and CCNA2 in breast cancer tissues of TNBC patients. Left, each box represents a patient. Right, correlation of their mRNA levels in the TNBC patients with indicated Pearson correlation coefficient. From cBioPortal. n = 82 patient samples. c,f, RNA immunoprecipitation-sequencing (RIP-seq) profiles for CCNA2 (c) and SMAD6 (f) based on our previous work . Blue, anti-FLAG RIP-seq in control or 293 cells expressing the FLAG-HA-tagged AKAP95 WT or mutants. Red, anti-AKAP95 RIP-seq in control or AKAP95-KD 293 cells. Black, profiles of total input RNAs. All profiles have the same Y-axis scale. Arrows indicate AKAP95-binding sites at intron 1. One representative RIP-seq analysis from 2 repeats. d, Total RNAs were used for RT-PCR, in the absence (- RT) or presence (+ RT) of reverse transcriptase, for CCNA2 intron 1 in MDA-MB-231 cells with indicated siRNAs. Top, PCR products on agarose gel. Asterisk, an unknown amplification product. Repeated 3 times. Bottom, relative ratios of the signal for the intron 1- retaining transcript over the intron 1-spliced transcript, as mean ± SD from n = 3 independent experiments. e, Assay for CCNA2 mRNA stability. Control (Scr) and AKAP95-KD MDA-MB-231 cells were treated starting from 0 min with Actinomycin D (+A, to block RNA synthesis) and cycloheximide (+C, to block NMD) or not as indicated. Total RNA at indicated times were used for RT-PCR and normalized to ACTB , as mean ± SD from n = 3 biological repeats. g, mRNA-seq profiles for SMAD6 in MDA-MB-231 cells expressing control or AKAP95 shRNA #1 (KD) and vector or AKAP95-expressing construct. Asterisk, a stop codon. P values by two-sided Student’s t -test for a and e and one-way ANOVA followed by Tukey’s post hoc test for d. Uncropped blots and statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Over Expression, Immunoprecipitation, Sequencing, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Blocking Assay, shRNA, Plasmid Preparation, Construct

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a,b, MDA-MB-231 cells virally expressing control or indicated shRNAs ( a ) or shRNA combined with indicated constructs ( b ) were subject to immunoblotting of total cell lysates (top) and colony formation assay. Middle, colony numbers as mean ± SD from n = 3 ( a ) or mean from 2 ( b ) independent experiments. Bottom, images of cells stained with crystal violet. c, Top, mRNA-seq profiles for CCNA2 in control or AKAP95 KD 293 cells . The numbers of exon junction reads are indicated. The red asterisk at the gene diagram indicates a stop codon 57 bp downstream of exon 1 in the intron. The number of reads for the junction of exons 1 and 2, and for the average neighboring exons, and their ratios are in the tables below for indicated cells. d, Total RNAs were used for RT-PCR for intron 1 region in control and AKAP95-KD MDA-MB-231 cells treated with or without cycloheximide for 6 hours. Repeated 3 times. e, Relative mRNA levels of UPF in UPF1-KD samples and BTZ in BTZ-KD samples, respectively, each relative to the control samples, as determined by RT-qPCR and normalized to GAPDH . Mean is from 2 independent experiments. f, Relative expression level of TGF-β pathway genes based on RNA-seq reads from control and AKAP95-KD MDA-MB-231 cells expressing vector or AKAP95. Venn diagram shows numbers of TGF-β pathway genes (from GSEA) downregulated by AKAP95 KD and upregulated by rescue with AKAP95 expression, and the relative expression of the 16 overlapped genes in both categories are plotted. One representative analysis from 2 repeats. g, RIP-seq profiles showing AKAP95 binding to RPUSD3 and PPM1K pre-mRNAs. Track information is the same as in . Red circles indicate the alternatively included exons (corresponding to the middle exon in the gene diagrams in ( h ), and red boxes show AKAP95 binding at the introns flanking these exons. h, Sashimi plots showing that the alternative splicing of RPUSD3 and PPM1K pre-mRNAs was affected by AKAP95 KD and rescued by restored expression of AKAP95. The numbers of exon junction reads and PSI are indicated. P values by two-sided Student’s t -test for a and b. Uncropped blots and statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Expressing, shRNA, Construct, Western Blot, Colony Assay, Staining, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, RNA Sequencing Assay, Plasmid Preparation, Binding Assay

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, Schematic of the “Knockout-first” Akap95 allele (null without further recombination). b, Body weights of mice with indicated Akap95 genotypes. Not significant (n.s.) between any two groups at any time for male, or after week 26 for female. *between Het and KO before week 26 for female. c, Peripheral blood profiles of 8-week mice of indicated genotype. n in b, c refers to the number of mice analyzed. d, MEFs were derived from mouse embryos. Images of six embryos from the same litter were shown at the top, followed with genotyping results, Ponceau S staining, and immunoblotting of MEFs. e, Top, heatmap showing relative expression levels of genes and clustered by changes in un-transduced KO MEFs from 2 embryos each. It includes 203 and 20 genes down- or up-regulated in KO, respectively. Bottom, heatmap showing relative alternative splicing of genes clustered by PSI changes. It includes 285 and 332 alternative splicing events with decreased or increased PSI in KO, respectively. Also see and . f, Top 10 gene sets enriched in genes down- (left, n = 265 genes) and up- (right, n = 742 genes) regulated in the MYC-transduced KO versus Het MEFs. g, Rescue of the gene expression profile by introduction of human AKAP95 into the MYC-transduced KO MEFs as shown by immunoblotting and heatmap for relative expression of down- or up-regulated genes in the indicated cells. Also see . Repeated 2 times. h, Relative expression of Akap95 and Akap8l in un-transduced (-MYC) and MYC-transduced (+MYC) MEFs from n = 2 Het and two KO embryos, as determined by normalized RNA-seq reads. i, Heatmap showing relative alternative splicing of genes clustered by PSI changes in MYC-transduced MEFs from 2 embryos each. It includes 216 and 252 alternative splicing events with decreased or increased PSI in KO versus Het MEFs, respectively. Also see . j, Gene ontology analysis for the indicated gene clusters from the heatmap in i . Blue (n = 216 genes) and red (n = 252 genes) show functions significantly enriched in genes with PSI increase or decrease by KO, respectively. k, Sashimi plots showing alternative splicing changes for each gene cluster from the heatmap using two examples, Asb7 for cluster 1, and Xpo4 for cluster 2. ns, not significant, *P < 0.05, by two-sided Student’s t -test for b, one-way ANOVA for c and modified Fisher’s exact test for f, j. Uncropped blots and statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Knock-Out, Derivative Assay, Staining, Western Blot, Expressing, RNA Sequencing Assay, Modification

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, Growth of MEFs from Akap95 +/- (Het) and Akap95 −/− (KO) embryos (n = 6 embryos each). b, Relative HRAS and MYC mRNA levels by RT-qPCR and normalized to Actb , as mean from HRAS G12V and MYC transduced MEFs (2 embryos each). c, HRAS-MYC-transduced MEFs in colony formation assay. Colony numbers as mean ± SD from n = 6 experiments using MEFs of 2 embryos each. d, Six mice received HRAS-MYC-transduced Het and KO MEFs on each flank. Tumor weights (week 4) are plotted. Each dot represents a tumor. e-i, MYC-transduced MEFs from 3 KO and 3 Akap95-expressing (1 WT, 2 Het) embryos. e, Right, images of cells before and after MYC transduction. Images of SA-beta-galactosidase activity assay are at bottom. Relative MYC mRNA levels after transduction were determined by RT-qPCR and normalized to Actb (left top). Percentage of SA-beta-gal-positive cells are plotted (left bottom). Both as mean ± SD from MEFs (n = 3 embryos each). f, Heatmap showing relative expression of genes and clustered by changes in KO MEFs (2 embryos each separately analyzed), with 265 and 742 genes down- or up-regulated in KO, respectively. Also see . g, Gene ontology analysis for the indicated gene clusters from the heatmap in f. Blue (n = 265 genes) and red (n = 742 genes) show functions significantly enriched in down- and up-regulated genes, respectively. h, GSEA plots above and below dashed line show gene sets significantly enriched in genes down- and up-regulated in the MYC-transduced KO compared to Het MEFs, respectively. i, Relative Akap95 and Ccna2 mRNA levels before and after MYC transduction by RT-qPCR and normalized to Actb , as mean ± SD from MEFs from n = 3 embryos each. j, Diagram summarizing regulation of tumorigenesis by AKAP95 through gene expression control. P values by two-sided Student’s t -test for all except one-way ANOVA followed by Tukey’s post hoc test for i, and modified Fisher’s exact test for g. Statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Quantitative RT-PCR, Colony Assay, Expressing, Transduction, Activity Assay, Modification

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, Immunoblotting for AKAP95 in HeLa cell nuclear extract and AKAP95 immunoprecipitation from the extract. Samples were boiled in the presence of DTT and resolved by SDS-PAGE. b, Disorder plot of human AKAP95. c, Turbidity by pictures and OD600 of MBP (none) and MBP fused to AKAP95 truncations at indicated concentrations all in 30 mM NaCl before and after TEV protease treatment for indicated time. OD600 is plotted as mean ± SD from n = 3 biological repeats. d, DIC (top) and fluorescence microscopy (bottom) images for 20 μM MBP-AKAP95 (101–210) and spiked with Oregon-green-labeled same protein (molar ratio 10:1) after TEV protease treatment for 30 min. Changes in NaCl concentration is indicated. Images were taken 5 min after salt adjustment. e, Phase contrast images of 50 μM MBP-AKAP95 (101–210) in 30 mM NaCl in the absence and presence of 10% of PEG6000 after TEV protease treatment for 30 min. f, Fusion of two droplets formed by 50 μM MBP-AKAP95 (101–210) in 30 mM NaCl and 10% of PEG6000 after TEV protease treatment for 30 min. Also see . g, DIC and fluorescence microscopy images of 6.25 μM MBP, MBP fused to Δ(101–210) or full-length AKAP95 in 150 mM NaCl, spiked with Oregon-green-labeled AKAP95 (101–210) at a molar ratio of 150:1 after TEV protease treatment for 30 min. Note that the lack of any condensates in the DIC images showed the inability of Δ(101–210) in condensation. Experiments in a, d, e-g were repeated 4 times. Scale bar, 5 μm for all. Uncropped blots and statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Western Blot, Immunoprecipitation, SDS Page, Fluorescence, Microscopy, Labeling, Concentration Assay

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, 293T cells were transfected with either empty vector (vec), or indicated AKAP95 construct with FLAG-HA-tag. Following α-Flag IP, the pulldown proteins were boiled and resolved by SDS-PAGE and detected by immunoblotting with α-HA. Blue and red asterisks indicate monomer and dimer, respectively. b, Identification of 1–100 as a probable prion subsequence on AKAP95. By the PLAAC program, using homo sapiens as background and core length of 30. c, Purified MBP and MBP fused to AKAP95 truncations as indicated or full-length AKAP95 (1–692) were resolved on SDS-PAGE and stained with Coomassie blue. d, MBP fused to AKAP95 truncations as indicated or full-length AKAP95 were resolved on SDS-PAGE and stained with Coomassie blue following treatment with TEV protease. Note that the cleaved MBP serves as a better indicator for cleavage efficiency as staining signal various for protein fragments of different sequences and sizes. e, Another event of fusion of two droplets formed by 50 μM MBP-AKAP95 (101–210) in 30 mM NaCl and 10% of PEG6000 after treatment with TEV protease for 30 min. Scale bar, 5 μm. Also see . f, Quantification of nuclear AKAP95 concentration by anti-AKAP95 Western blot. Total lysates from indicated number of MDA-MB-231 (M231) and flp-TREx 293 cells (f293, un-induced and dox-induced for FH-AKAP95 expression) were loaded, along with indicated ng of purified MBP-AKAP95. AKAP95 signal of un-induced f293 is similar to that of 25 ng of MBP-AKAP95. All experiments were repeated 2 times. Uncropped blots are provided as in source data .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Transfection, Plasmid Preparation, Construct, SDS Page, Western Blot, Purification, Staining, Concentration Assay, Expressing

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, Immunostaining of endogenous AKAP95 (red) and DNA (DAPI, blue) in indicated cancer cell lines and primary MEFs from WT and Akap95 KO embryos. b, Confocal microscopy images of AKAP95 WT or ZF C-S fused to GFP in nuclei following transfection into HeLa cells. c, Fluorescence microscopy images of HeLa cells transiently expressing AKAP95 WT or Δ(101–210 fused to GFP. d, HeLa cells were transfected with AKAP95-GFP, and two nuclei were imaged at different time points. Time 0 was 24 hr after transfection. Note the growth and merge of the foci, especially those in the red circle. e, Rapid fusion of AKAP95 (ZF C-S )-GFP foci in a HeLa cell nucleus. The white oval and arrow show two different fusion events. These images are from . All experiments were Repeated 4 times. Scale bar, 5 μm for all.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Immunostaining, Confocal Microscopy, Transfection, Fluorescence, Microscopy, Expressing

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: Fluorescence microscopy images of HeLa cells transiently expressing AKAP95-GFP. Nucleus DNA was stained by DAPI, and specific proteins were stained with antibodies for SRSF2 ( a ), Pol II ( b ), and Pol II-S2P ( c ). The assays were repeated 10 times for each staining and show similar trend. Right, quantification of the signal intensity of indicated molecules across the dotted lines shown in the images. Quantification by Image J. Scale bar, 5 μm for all. Statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Fluorescence, Microscopy, Expressing, Staining

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, Alignment of human and mouse AKAP95 (101–210). Middle row shows identical residues (letter) and conservative mutations (“+”). Tyr, red; Phe, blue and tall. Box, Tyr and Phe swapping. b, MBP alone (none) or MBP-AKAP95 (101–210) WT or mutants all at 50 μM and in 30 mM NaCl after TEV protease treatment for 30 min. Turbidity of each reaction was shown, and by OD600 as mean ± SD from n = 3 (for YA, YS) or 4 (the rest) independent assays. Samples taken after mixing and from supernatant after centrifugation were resolved by SDS-PAGE followed by coomassie blue staining. c, DIC and fluorescence microscopy images for 10 μM Oregon-green-labeled MBP-AKAP95 (101–210) WT and mutants in 30 mM NaCl after TEV protease treatment for 30 min. Plots from left to right show relative protein amount in droplet, number of droplets in a field, and ratio of protein concentration inside droplets over sum of inside and outside droplets, respectively, as mean ± SD from n = 24 randomly picked droplets, except for number of droplets from n = 3 randomly picked fields, in one representative assay from 5 repeats. NA, not applicable. d, Fluorescence microscopy images of HeLa cells (top) and Flp-In T-Rex 293 cell lines expressing (bottom) GFP fusions with full-length AKAP95 WT or mutants. Repeated 4 times. e,h, HEK293 cells co-transfected with indicated siRNAs and plasmids were subject to splice reporter assay (top) and immunoblotting with α-AKAP95 (bottom). Δ = Δ(101–210). Mean ± SD from n = 8 [except 5 for Δ(101–210) and 13 for YF and 2 nd WT] independent transfections are plotted in e and 7 independent transfections in h. f, Schematic of AKAP95 chimeras. g, Fluorescence microscopy images of 293T cells transfected with indicated AKAP95 chimeras fused to GFP. Repeated 3 times. P values by two-sided Student’s t -test for b and one-way ANOVA followed by Tukey’s post hoc test for e and h. Scale bar, 5 μm for all. Uncropped blots and statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Centrifugation, SDS Page, Staining, Fluorescence, Microscopy, Labeling, Protein Concentration, Expressing, Transfection, Reporter Assay, Western Blot

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, Amino acid enrichment for AKAP95 (101–210). By Composition Profiler, using SwissProt 51 Dataset as background. b, Purified MBP fused to AKAP95 (101–210) WT and mutants were resolved on SDS-PAGE and stained with Coomassie blue. Repeated 2 times. c, Disorder plot of AKAP95 WT or mutants with indicated mutations in 101–210. d, MBP-AKAP95 (101–210) WT, YS, and YF, all at 35 μM and in 30 mM NaCl, were treated with TEV protease for 2 hrs in 3 independent assays, and subjected to centrifugation. The supernatant and pellets (resuspended in the same volume as the supernatant) were resolved by SDS-PAGE followed with coomassie blue staining. MBP signal in the pellet reflects residual supernatant fraction, and its percentage [MBP pellet/(supernatant + pellet)] was subtracted from the (101–210) pellet percentage. Such normalized (101–210) pellet percentages are plotted as Partition Percentage as mean ± SD of n = 3 independent experiments. It is most likely that all supernatants may also have substantial portion of condensates. Moreover, the size cutoff of condensates is also arbitrary, as protein assemblies may take a continuum of size distribution . e, Immunoblotting by α-AKAP95 (top) or GAPDH (bottom) of total lysates from Flp-In T-Rex 293 cell lines induced to express full-length AKAP95 WT or indicated mutants fused to GFP. Repeated 3 times. f,g, Indicated full-length AKAP95 WT or mutants fused to GFP were induced by various concentrations of doxycycline in Flp-In T-Rex 293 cell lines. Immunoblotting of total cell lysates with indicated antibodies ( f ). The doxycycline concentrations in red font activated the transgene at the near endogenous level, and were selected for treating cells and fluorescence microscopy assays of fixed cells in ( g ). Scale bar, 5 μm. Repeated 2 times. h, 293T cells transiently expressing indicated constructs with FLAG-HA-tag were used for α-FLAG immunoprecipitation and immunoblotting with indicated antibodies and Ponceau S staining. Repeated 2 times. i, Immunoblotting of 293T cells transfected with empty vector or indicated FLAG-HA-tagged AKAP95 chimeras fused to GFP. Bottom, by anti-GAPDH. Top, by anti-AKAP95 (Bethyl Laboratories, A301–062A, recognizes an epitope in a region between residue 575 and 625 of human AKAP95). Repeated 2 times. P values by two-sided Student’s t -test for d. Uncropped blots and statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Purification, SDS Page, Staining, Centrifugation, Western Blot, Fluorescence, Microscopy, Expressing, Construct, Immunoprecipitation, Transfection, Plasmid Preparation

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, OD450 at different time after TEV protease treatment of 50 μM MBP-AKAP95 (101–210) WT, YS, and YF in 150 mM NaCl, as mean ± SD of readings after subtracting that of MBP at each time (constant at 0.07–0.08) from n = 3 independent assays. Dashed lines show half of the maximum turbidity and time (τ 1/2 ) to reach it. b, Ratio of protein concentration inside the droplets over sum of inside and outside for (101–210) WT and YF at increasing protein concentrations and in 30 mM NaCl, as mean ± SD from n = 6 randomly picked droplets each, in one representative assay from 3 repeats based on . c, Confocal microscopy images of GFP-AKAP95 (101–210) WT and YF at increasing protein concentrations, all in 150 mM NaCl and 10% of PEG6000 after TEV protease treatment for 20 min. Repeated 2 times with similar results. d, DIC and fluorescence microscopy images for 20 μM MBP-AKAP95 (101–210) YF spiked with Oregon-green-labeled same protein (molar ratio 10:1), after TEV protease treatment for 30 min. Changes in NaCl concentration is indicated. Images were taken 5 min after salt adjustment. Repeated 2 times with similar results. e, Different extent of droplet fusion (arrows) by AKAP95 (101–210) WT and YF, both at 50 μM and in 30 mM NaCl and 10% of PEG6000 after TEV protease treatment for 30 min. A similar trend was observed in 5 fusion events (or attempted fusion for YF) for each. Also see – . f, Equation for Line Raster Scan Image Correlation fitting autocorrelation G(Ψ), which depends on G(0)=γ/N (γ: beam profile, N = number of mobile particles), diffusion coefficient D (μm 2 /s), line scan time t l , pixel size Ψ, radial beam waist w 0 and axial beam waist w z . The radial waist w 0 (0.218 μm) was calibrated with sub-diffraction beads (0.1 μm) diluted solution as reported before . The axial waist w z was considered equal to 3*w 0 . g, Fluorescence confocal microscope image of HeLa cell expressing full-length AKAP95 WT or YF fused to GFP. Arrow indicates the scanned region for in vivo Line RICS. h, Fluorescence confocal microscope image of GFP-AKAP95 (101–210) WT and YF in 150 mM NaCl and 10% of PEG6000 after TEV protease treatment for 20 min, for in vitro Line RICS experiments. i, Line Fluorescence carpet formed by ~10 4 lines (each line is composed by 128 pixels, 50 nm/pixel) acquired with 0.101 s line scan time and 32.8 μs/pixel. Line RICS autocorrelation curves were computed on 64 sections of 128 lines followed by averaging all the curves. The sectioning of the line carpet permitted to avoid the effect of the movement of the condensates on the measurement of the autocorrelation curves. (λex=488 nm). j, Line RICS autocorrelation curves for experimental data and fitted. k, Residuals of the fitting. Experiments in g-i were repeated 3 times with similar results. Scale bar, 2 μm for c and h, 5 μm for d, e, and g.

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Protein Concentration, Confocal Microscopy, Fluorescence, Microscopy, Labeling, Concentration Assay, Diffusion-based Assay, Expressing, In Vivo, In Vitro

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a, Fluorescence microscopy images of Oregon-green-labeled AKAP95 (101–210) WT and YF at indicated protein and NaCl concentrations after MBP cleavage for 30 min. Repeated 3 times. b, Fluorescence microscopy images of 50 μM AKAP95 (101–210) WT and YF in 30 mM NaCl both spiked with Oregon-green-labeled (101–210) WT (molar ratio 150:1) after MBP cleavage for 30’ and imaged immediately (30’) or after incubation for 60 (90’) or 120 (150’) more minutes. Repeated 3 times. c, FRAP of 10 μM GFP-AKAP95 (101–210) WT and YF after 30 min of MBP cleavage in 150 mM NaCl. FRAP was performed immediately (30’) or after incubation for 60 min more (90’). Left, fluorescence microscopy images of droplets at indicated times. Middle, FRAP recovery curves as mean ± SD. Right, mean ± SD of recovery at the final time. n = 7 independent measurements. d, FRAP of Full-length AKAP95 WT and YF fused to GFP in HeLa cell nuclei. Left, fluorescence microscopy images of foci. The photobleached focus was boxed and amplified for indicated time points. Middle, FRAP recovery curves as mean ± SD. Right, mean ± SD of recovery (relative to minimal level) at the final time. n = 7 independent measurements. e,f, Diffusion of full length AKAP95 WT and YF fused to GFP in HeLa cell nuclei, showing Line RICS normalized autocorrelation curves G(Ψ) as function of Spatial Lag (Ψ) ( e ) and diffusion coefficients ( f ), as mean ± SD (n = 20 or 22 independent measurements for WT, YF, respectively). g,h, Diffusion coefficients of purified GFP-AKAP95 (101–210) WT and YF, showing Line RICS normalized autocorrelation curves G(Ψ) ( g ) and diffusion coefficients ( h ), as mean ± SD (n = 22 or 13 independent measurements for WT, YF, respectively). Scale bars, 10 (a, b), 2 (c), 5 (d) μm. P values by two-sided Student’s t -test (c, d) or Mann-Whitney U test (f, h). For box-and-whisker plots, data are median (line), 25–75th percentiles (box) and minimum-maximum values recorded (whiskers). Statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Fluorescence, Microscopy, Labeling, Incubation, Amplification, Diffusion-based Assay, Purification, MANN-WHITNEY, Whisker Assay

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a-f, MDA-MB-231 transduced with control or AKAP95 shRNA #1 (KD) and vector or FLAG-HA-tagged full-length AKAP95 WT or mutants. n = 3 biological repeats for a-f, except n = 4 biological repeats for e. a, Immunoblotting of total cell lysates. b, Colony formation assays. Left, colony numbers as mean ± SD. Right, images of cells stained with crystal violet. c, Growth of cultured cells, as mean ± SD. d, Relative SMAD6 mRNA level were determined by RT-qPCR and normalized to GAPDH , as mean ± SD. e,f, RT-PCR for ratios for intron 1-retained over -spliced CCNA2 (e) and exon-included over -skipped RPUSD3 transcripts, as mean ± SD. g-k, MYC-transduced Akap95 KO MEFs transduced with vector or HA-tagged full-length AKAP95 WT or mutants. g, Immunoblotting of total cell lysates. Repeated 3 times. h, Left, percentage of SA-beta-gal-positive cells as mean ± SD (n = 3 different images of MEFs from two embryos). Right, images from KO MEF 1. i, Relative mRNA levels of indicated genes with related functions at bottom by RT-qPCR and normalized to Gapdh , as mean ± SD (n = 3 independent experiments). * or ** between vec and WT, WT and YS, WT and YF, except for Plk1, for which * only between vec and WT, WT and YF. *P<0.05, **P<0.01. j, Heatmap showing relative alternative splicing with PSI changes in MYC-transduced KO MEFs expressing indicated constructs (2 embryos each). Also see . k, Sashimi plot showing Aamdc alternative splicing that was rescued by introduction of AKAP95 WT, but not but the mutant, and RT-PCR for the inclusion of the alternative exon as mean ± SD (n = 4 biological replicates pooled from 2 embryos each). l, Diagram showing impact of material properties of AKAP95 WT and mutants on gene regulation and tumorigenesis. P values by two-sided Student’s t -test for c and one-way ANOVA followed by Tukey’s post hoc test for all other analyses. Uncropped blots and statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Transduction, shRNA, Plasmid Preparation, Western Blot, Staining, Cell Culture, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Expressing, Construct, Mutagenesis

Journal: Nature cell biology

Article Title: Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis

doi: 10.1038/s41556-020-0550-8

Figure Lengend Snippet: a,b, MDA-MB-231 cells were virally infected to stably express scramble (control) or AKAP95 shRNA #1 (KD) and the indicated constructs including empty vector (vec) and FLAG-HA-tagged full-length AKAP95 WT or mutants. a, Relative CCNA2 mRNA level as determined by RT-qPCR and normalized to GAPDH , and plotted for each of the 2 biological repeats individually. b, RT-PCR for ratios for exon-included over -skipped PPM1K transcript, as mean ± SD from n = 3 biological repeats. c-f, MYC-transduced Akap95 KO MEFs were transduced with vector or constructs expressing HA-tagged full-length AKAP95 WT or mutants. c, Heatmap showing relative expression levels of genes changed in MYC-transduced KO MEFs (from 2 embryos each) stably expressing indicated rescue constructs. Also see . d, Relative mRNA levels of indicated SASP genes as determined by RNA-seq reads from 2 biological repeats (KO1 and KO2). e,f, Sashimi plots showing example genes for which the alternative exon inclusion was promoted ( e ) or suppressed ( f ) by introduction of AKAP95 WT, but not as effectively by YS or YF, and RT-PCR for the inclusion of the alternative exon, as mean from 2 embryos each. g, A model for how AKAP95 condensates may regulate gene expression for tumorigenesis. P values by one-way ANOVA followed by Tukey’s post hoc test. Uncropped blots and statistical source data are provided as in .

Article Snippet: Rabbit polyclonal anti-AKAP95, Santa Cruz Biotechnology Cat#sc-10766; Rabbit polyclonal anti-AKAP95, Bethyl Laboratories Cat#A301–062A, Mouse monoclonal anti-GAPDH, EMD Millipore Cat#MAB374; AlexaFluor 555 conjugated goat anti-rabbit IgG, Thermo Fisher Scientific Cat#A-21428; Mouse anti-SRSF2 antibody, Abcam Cat#Ab11826; mouse anti-Pol II, 8WG16 clone, COVANCE Cat# MPY-127R; Mouse anti-pol II CTD-S2P, H5 clone, COVANCE Cat#MPYT-127R; Rabbit polyclonal anti-cyclin A, Santa Cruz Biotechnology Cat#sc-751; Rabbit polyclonal anti-DDX5, Santa Cruz Biotechnology Cat#sc-32858; Mouse monoclonal anti-hnRNP M, Santa Cruz Biotechnology Cat#sc-20002; AlexaFluor 555 conjugated goat anti-mouse IgG, Thermo Fisher Scientific Cat# A-21422.

Techniques: Infection, Stable Transfection, shRNA, Construct, Plasmid Preparation, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Transduction, Expressing, RNA Sequencing Assay

Journal: Nature genetics

Article Title: BRD2 Compartmentalizes the Accessible Genome

doi: 10.1038/s41588-022-01044-9

Figure Lengend Snippet: (a) Enrichment profile and heatmap of BRD2/3/4, CTCF, P300 ChIP-seq signal at their respective binding peaks before and after BRD4 depletion. (b) Representative genomic tracks (from integrated genomics viewer) of ChIP-seq signal of BRD2, BRD4 or CTCF after acute depletion of BRD4 for 6 hours. (c) Representative single cell view of Cohesin (RAD21, green), BRD2 (cyan) and BRD4 (magenta) and merged BRD2/BRD4. The cyan arrow indicates the BRD2 puncta showing little colocalization with BRD4. The magenta arrow indicates the BRD4 puncta poorly colocalized with BRD2. The white line indicates the region to profile the fluorescent intensity. Scale bar, 1μm. (d) Fluorescence intensity profile of BRD2(cyan) and BRD4 (magenta) along the while line in (c). The relative intensity ratio is plotted. (e) WB analysis of the Rad21-AID : Brd4-dTAG : Brd2-dTAG triple edited ESC line. dTAG13 treatment (100nM, 6 hours) simultaneously depletes both BRD2 and BRD4 whereas auxin treatment (100μM, 6 hours) orthogonally depletes RAD21. (f) Histogram of eigenvector values from the Pearson’s correlation matrix of single RAD21 depletion, dual RAD21/BRD4 or triple RAD21/BRD4/BRD2 depletion for 6 hours compared to untreated Control from Micro-C experiments. (g) Quantification of the digitalized A-A compartmental interactions (log10 value of observed/expected) after single RAD21 depletion, dual RAD21/BRD4 depletion or triple RAD21/BRD2/BRD4 depletion for 6 hours from Micro-C experiments. Triple RAD21/BRD4/BRD2 depletion reduced the enhanced compartmentalization after the dual Cohesin/BRD4 depletion. The black solid line in each violin plot represents the median value. (h) Degradation of BRD2 significantly reduced the accessible chromatin clustering after dual BRD4/RAD21 depletion. g(r) curves were plotted for indicated conditions. The non-parametric two-sided Mann-Whitney U test was used for statistical testing. (i) Biallelic knock-in of HaloTag into endogenous BET family genes enable accurate quantification of individual BET family protein copy number by CTCF-calibrated flow cytometry in live cells. The mean and standard deviation of the quantified copy number from two biological experiments are shown above each plot. The non-parametric Mann-Whitney U test was used for statistical testing. ***, p < 0.001; n.s., not significant.

Article Snippet: The dTAG constructs was derived from pCRIS-PITChv2-dTAG-Puro (BRD4) (Addgene, 91796) and donor constructs for Brd2/3/4 were constructed by PCR amplification and Gibson assembly.

Techniques: ChIP-sequencing, Binding Assay, Fluorescence, Control, MANN-WHITNEY, Knock-In, Flow Cytometry, Standard Deviation

Journal: Nature genetics

Article Title: BRD2 Compartmentalizes the Accessible Genome

doi: 10.1038/s41588-022-01044-9

Figure Lengend Snippet: (a) A schematic of the dual targeted protein degradation strategy. In Rad21-eGFP-mAID cells, a FKBP F36V based degron (dTAG) linked to a HaloTag was bi-allelically knocked into endogenous BET family genes ( Brd2, Brd3 and Brd4 ) by the CRISPR/Cas9 genome editing method (only one engineered allele is shown). Adding cell membrane permeable dTAG13 ligand into the culture will bring the dTAG labeled BET proteins into close proximity to the Cereblon (CRBN) E3 ligase for proteasome-mediated protein degradation orthogonal to the mAID system. (b) Western blot (WB) analysis of protein levels of endogenous BRD2-dTAG or RAD21-AID individually or in combination after 6 hours of dTAG13 or auxin treatment, respectively. Rapid depletion of BRD2 or RAD21 individually or together does not impact the protein level of other BET proteins (BRD3 or BRD4). (c) Degradation of BRD2 for 6 hours significantly reduced the accessible chromatin clustering after RAD21 depletion. g(r) curves were plotted for indicated conditions. (d-e) Violin plot of 3D volumes (the number of voxels) of two ATAC-rich segments R2 ( d ) and R5 ( e ) before and after BRD2 depletion, RAD21 depletion or in combination. The black bar indicates the median value for each data set and Mann-Whitney U test was performed for the statistical analysis. The number of analyzed alleles for the ATAC-rich segment (Chr4-R2) are : Control (n=93), RAD21 depletion (n=129), BRD2 depletion (n=116), or dual depletion of both RAD21 and BRD2 (n=119). The number of analyzed alleles for the ATAC-rich segment (Chr4-R5) are : Control (n=91), RAD21 depletion (n= 85), BRD2 depletion (n=99), or dual depletion of both RAD21 and BRD2 (n=121). (f) Pearson’s correlation matrix of the whole chromosome 17 for BRD2 or RAD21 depletion alone or in combination for 6 hours from Micro-C experiments. Dual BRD2 and RAD21 depletion reduced the enhanced compartmentalization after Cohesin depletion alone. (g ) Quantification of the digitalized A-A compartmental interactions (log10 value of observed/expected) after BRD2 or RAD21 depletion alone or in combination from Micro-C experiments. The black solid line in each violin plot represents the median value. (h-i) Differential saddle plot analysis (h) and quantitative A-A compartmental interactions (log10 value of observed/expected) analysis (i) by Micro-C for cells stably expressing empty vector, full length (FL), N-terminal double bromodomain (BD) and C-terminal low complexity domain (LCD) of BRD2. The non-parametric two-sided Mann-Whitney U test was used for statistical testing. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Article Snippet: The dTAG constructs was derived from pCRIS-PITChv2-dTAG-Puro (BRD4) (Addgene, 91796) and donor constructs for Brd2/3/4 were constructed by PCR amplification and Gibson assembly.

Techniques: CRISPR, Membrane, Labeling, Western Blot, MANN-WHITNEY, Control, Stable Transfection, Expressing, Plasmid Preparation

Journal: Nature genetics

Article Title: BRD2 Compartmentalizes the Accessible Genome

doi: 10.1038/s41588-022-01044-9

Figure Lengend Snippet: (a ) WB test of time and dose-dependent degradation of protein levels of endogenous BRD2-dTAG. 100nM dTAG13 is sufficient to deplete BRD2 as early as 3 hours. We used 100nM dTAG13 treatment for 6 hours throughout the current study. (b-c ) Quantification of single cell fluorescence of endogenously labeled HaloTag-BRD2/3/4-using HaloTag ligand JF 646 before and after dTAG13 treatment for 6 hours (b). RAD21 GFP signal serves as the control (c). (d-f ) Representative images of acute degradation of BRD2 ( d ), BRD3 ( e ) and BRD4 ( f ) after 6 hours of dTAG13 treatment. Endogenous BET proteins were engineered with a HaloTag and labeled with 500nM JF 646 . The RAD21 level was monitored by GFP. Scale bar,5 μm. (g-i) Cell cycle analysis after 6 hours depletion of BRD2, BRD3 or BRD4 by propidium iodide staining. Histograms of the DNA content distribution measured by propidium iodide staining are shown. (j ) WB analysis of protein levels of endogenous BRD3-dTAG or RAD21-AID individually or in combination after 6 hours of dTAG13 or auxin treatment, respectively. (k ) Depletion of BRD3 for 6 hours did not significantly reduce the accessible chromatin clustering after RAD21 depletion. g(r) curves were plotted for indicated conditions. The two-sided Mann-Whitney U test was used for statistical testing. (l) Violin plot of 3D volumes (the number of voxels) of two ATAC-rich segments R2 (left) and R5 (right) before (grey) and after (red) BRD3 depletion, RAD21 depletion or in combination. The number of alleles analyzed is indicated at the bottom. The black bar indicates the median value for each data set and Mann-Whitney U test was performed for the statistical analysis. n.s.,not significant. (m) WB analysis of protein levels of endogenous BRD4-dTAG or RAD21-AID individually or in combination after 6 hours of dTAG13 or auxin treatment, respectively. (n ) Degradation of BRD4 for 6 hours did not significantly reduce but instead had a trend to increase the accessible chromatin clustering after RAD21 depletion. g(r) curves were plotted for indicated conditions. The two-sided Mann-Whitney U test was used for statistical testing. (o) Violin plot of 3D volumes (the number of voxels) of two ATAC-rich segments R2 (left) and R5 (right) before (grey) and after (red) BRD4 depletion, RAD21 depletion or in combination. The number of alleles analyzed is indicated at the bottom. The black bar indicates the median value for each data set and Mann-Whitney U test was performed for the statistical analysis.

Article Snippet: The dTAG constructs was derived from pCRIS-PITChv2-dTAG-Puro (BRD4) (Addgene, 91796) and donor constructs for Brd2/3/4 were constructed by PCR amplification and Gibson assembly.

Techniques: Fluorescence, Labeling, Control, Cell Cycle Assay, Staining, MANN-WHITNEY

Journal: Nature genetics

Article Title: BRD2 Compartmentalizes the Accessible Genome

doi: 10.1038/s41588-022-01044-9

Figure Lengend Snippet: (a) Histogram of eigenvector values from the Pearson’s correlation matrix of single BRD2 or RAD21 depletion and dual RAD21/BRD2 depletion for 6 hours compared to untreated Control from Micro-C experiments. BRD2 depletion increases the switching of B to A compartments. (b ) Browser track view of the eigenvector values for compartmental scores in various perturbation conditions in chromosomes 11 (Chr11). The genomic regions containing B to A switches after BRD2 depletion (green line) are highlighted with black arrows. (c) Live cell single molecule tracking (SMT) of BET proteins by stroboscopic imaging. The jump length fitting of BRD2 dynamics is best described by a three-state model: diffusive, slow (likely transient, non-specific collision) and bound (likely stably bound to cognate sites). The probability distribution function of jump length or single molecule displacement was fit over multiple camera integration time scales. The same three-state model applies to BRD3 or BRD4 (data not shown). (d-e ) The chromatin bound fraction (d) and diffusion coefficient D (e) of BRD2, BRD3 and BRD4 before and after 6 hours of RAD21 depletion were quantified from SMT experiments. The number of cells analyzed are n=17 and n=19 for control and RAD21 depletion for BRD2 SMT, n=17 and n=17 for control and RAD21 depletion for BRD3 SMT, and n=17 and n=19 for control and RAD21 depletion for BRD4 SMT. (f) ChIP-seq analysis of BRD2, BRD3 and BRD4 after 6 hours Cohesin depletion. Shown are the enrichment profile (upper panel) and heatmap (lower panel) of each protein over its binding peaks. (g ) A violin plot showing the log2 fold change of BRD2 CUT&Tag and BRD2/3/4 ChIP-seq intensity at corresponding peaks. Both BRD2 CUT&Tag and BRD2 ChIP-seq show preferential increase at BRD2 ChIP-seq peak regions compared to BRD3/BRD4. (h) Putative model of B to A compartmental switch after removing BRD2. The active A compartment is colored in pink whereas the inactive B compartment in cyan. BRD2 molecules bind to the active A compartment enriched with CTCF and acetylated nucleosomes. BRD2 depletion weakens the boundary resulting in more Cohesin translocation into the neighboring inactive segments and more A/B mixing. (i) Putative model showing the enhanced chromatin binding of BRD2 associated with an increased spatial clustering of ACDs upon Cohesin loss. For simplicity, two small segments containing acetylated nucleosomes are shown. The non-parametric Mann-Whitney U test was used for statistical testing. **, p < 0.01; n.s., not significant.

Article Snippet: The dTAG constructs was derived from pCRIS-PITChv2-dTAG-Puro (BRD4) (Addgene, 91796) and donor constructs for Brd2/3/4 were constructed by PCR amplification and Gibson assembly.

Techniques: Control, Imaging, Stable Transfection, Diffusion-based Assay, ChIP-sequencing, Binding Assay, Translocation Assay, MANN-WHITNEY

Journal: Nature genetics

Article Title: BRD2 Compartmentalizes the Accessible Genome

doi: 10.1038/s41588-022-01044-9

Figure Lengend Snippet: (a) Quantification of compartmental changes after BRD2 and RAD21 depletion individually or in combination. A, active compartments; I, intermediated mixed compartments; B, inactive compartments. The mouse genome is binned into 25480 segments (1Mbp bin size) and the percentage of each fraction is shown. See details of in the section. (b ) Browser track view of the eigenvector values for compartmental score in various perturbation conditions on chromosome 17. The genomic regions containing B to A switches after BRD2 depletion (green line) are highlighted with black arrows. (c ) Endogenous Co-immunoprecipitation between BRD2 and CTCF in mouse ESCs. Benzonase (BZ) treatment did not abrogate the BRD2-CTCF interaction as compared to untreated control (UT). (d ) (Upper panel) Schematic of genome engineering of BET proteins with HaloTag and labeled with Janelia photoactivatable fluorophore PA-JF 549 for single molecule tracking. (Lower panel) Illustration of stroboscopic single molecule imaging of BET protein dynamics in live cells. After activation by a 405 nm pulse, single BET molecules were excited by 561 nm laser pulse (2 ms) to suppress motion blurring and images were captured with ~5ms exposure times. (e ) The probability distribution function of jump length was fit over multiple camera integration time scales by the two-state model estimated by Spot-On program (See ). The two-state diffusive vs bound model does not well account for the dynamics of BRD2 or BRD3/4 (not shown) as compared to the three-state model in . (f-g ) The diffusive fraction ( f ) and its diffusion coefficient ( g ) analysis for BRD2, BRD3 and BRD4 from SMT analysis after Cohesin depletion. Cohesin depletion significantly decreased the diffusive fraction of BRD2 accompanied with an increase of the chromatin bound fraction. The number of cells analyzed are the same as - . (h-i ) The slow bound fraction ( h ) and its diffusion coefficient ( i ) analysis for BRD2, BRD3 and BRD4 from SMT analysis after Cohesin depletion. Cohesin depletion also significantly decreased the diffusion coefficient of slow bound BRD2. The number of cells analyzed are the same as - .

Article Snippet: The dTAG constructs was derived from pCRIS-PITChv2-dTAG-Puro (BRD4) (Addgene, 91796) and donor constructs for Brd2/3/4 were constructed by PCR amplification and Gibson assembly.

Techniques: Immunoprecipitation, Control, Labeling, Imaging, Activation Assay, Diffusion-based Assay

Journal: Nature genetics

Article Title: BRD2 Compartmentalizes the Accessible Genome

doi: 10.1038/s41588-022-01044-9

Figure Lengend Snippet: (a ) Scatter plot of two biological replicates of ChIP-seq signal for BRD2, BRD3 and BRD4. Ranked spearman correlation coefficient is shown above each plot. (b ) Pie diagram of different genomic binding features of BRD2, BRD3 and BRD4 extracted from their respective ChIP-seq peak signal. The percentage of BET proteins binding to different genome features are summarized in the table on the bottom. (c) Enrichment of BET protein ChIP-seq signal at CTCF sites was performed using k-means clustering (n=2, enriched and non-enriched). BRD2 has the highest enrichment profile among BET proteins at CTCF sites at the binding-enriched cluster. (d) (Left panel) Representative genome browser track of Cohesin ChIP-seq (magenta) and BRD2 CUT&Tag (blue) before and after Cohesin depletion (RAD21(−)). Shaded area demonstrates the increased BRD2 signal at Cohesin binding peaks. (Right panel) BRD2 enrichment and heatmap analysis by CUT&Tag (Cell Signaling, 5848S) at Cohesin ChIP-seq peaks before and after Cohesin depletion. (e) Genome wide heatmap analysis of BRD2 CUT&Tag enrichment at Cohesin ChIP-seq peaks before and after Cohesin depletion by using a different antibody (Bethyl laboratories, A302-583A). (f) ChIP-seq analysis of RAD21 and CTCF after 6 hours Cohesin depletion. Shown are the enrichment profile (upper panel) and heatmap (lower panel) of each protein over its binding peaks. (g ) Chromatin fractionation assay of BRD2 and BRD4 in non-treated Control and RAD21 depletion conditions after different salt extraction of ESC nuclei followed by western blot analysis. GAPDH and Histone H4 was used as marker for the cytoplasmic and chromatin bound fraction, respectively. In control cells, BRD2 is preferentially extracted from the 300mM NaCl concentration. After Cohesin depletion, more BRD2 is extracted from the 600mM NaCl concentration. (h ) Summary of the relative BRD2 enrichment in different chromatin fractionations before and after RAD21 depletion from 4 replicates. Each fraction is normalized to histone H4 abundance. * indicates p < 0.05 by non-parametric Mann-Whitney test. Error bar represents standard deviation. The non-parametric Mann-Whitney U test was used for statistical testing. *, p < 0.05.

Article Snippet: The dTAG constructs was derived from pCRIS-PITChv2-dTAG-Puro (BRD4) (Addgene, 91796) and donor constructs for Brd2/3/4 were constructed by PCR amplification and Gibson assembly.

Techniques: ChIP-sequencing, Binding Assay, Genome Wide, Fractionation, Control, Extraction, Western Blot, Marker, Concentration Assay, MANN-WHITNEY, Standard Deviation

Journal: Nature genetics

Article Title: BRD2 Compartmentalizes the Accessible Genome

doi: 10.1038/s41588-022-01044-9

Figure Lengend Snippet: (a) Enrichment profile and heatmap of BRD2/3/4, CTCF, P300 ChIP-seq signal at their respective binding peaks before and after BRD2 depletion. (b) Enrichment profile and heatmap of ChIP-seq signal of BRD2, BRD3, BRD4 and CTCF at their cognate binding peaks after 6 hours of BRD3 depletion. (c) Heatmap of BRD2 binding signal specifically at BRD2/BRD4 co-bound regions before and after acute BRD4 depletion for 6 hours. (d ) Representative genomic tracks (from integrated genomics viewer) of IgG, BRD3 and p300 after BRD4 depletion. BRD4 depletion markedly reduced P300 binding. (e) Validating the specificity of fluorescence signal or antibodies used for immunofluorescence after acute depletion of RAD21 or BRD2/BRD4. The RAD21-mAID-eGFP : BRD2-HA-dTAG : BRD4-Halo-dTAG triple degron mESC line was used in this study. RAD21 was detected by covalently tagged eGFP signal. BRD2 was detected by mouse monoclonal anti-HA antibody and goat-anti-mouse Alexa568 secondary antibody. BRD4 was detected by rabbit BRD4 primary antibody and goat-anti-rabbit Alexa647 secondary antibody. To validate the fluorescence signal specificity, RAD21 and BRD2/BRD4 were depleted by adding auxin (500μM) or dTAG13 (100nM) for 6 hours, respectively. (f) (Upper panel) Representative images of spatial distribution of RAD21, BRD2 and BRD4 signal. (Lower panel) Fluorescent Intensity profile of RAD21(green), BRD2 (cyan) and BRD4 (magenta) relative to DAPI (blue). RAD21 is enriched at both DAPI high and low regions whereas BRD2 and BRD4 are enriched at DAPI low regions. Scale bar, 3μm. (g) Representative single cell view of Cohesin (RAD21, green), BRD2 (cyan) and BRD4 (magenta) in their merged view. The while line indicates the cross-sectional line to analyze the fluorescent intensity profile. The cyan arrow indicates the BRD2 puncta showing little colocalization with BRD4. The magenta arrow indicates the BRD4 puncta poorly colocalized with BRD2. Scale bar, 1μm. (h) Fluorescence intensity profile of Cohesin (RAD21, green), BRD2(cyan) and BRD4 (magenta) along the while line in (g). (i) Whole nucleus 3D voxel-to-voxel correlation summary of RAD21 vs BRD2 and BRD4 vs BRD2 as shown in . The percentage of voxel colocalization of protein pair, the Pearson’s coefficient, and the Manders’ coefficient are computed and summarized.

Article Snippet: The dTAG constructs was derived from pCRIS-PITChv2-dTAG-Puro (BRD4) (Addgene, 91796) and donor constructs for Brd2/3/4 were constructed by PCR amplification and Gibson assembly.

Techniques: ChIP-sequencing, Binding Assay, Fluorescence, Immunofluorescence

Journal: Nature genetics

Article Title: BRD2 Compartmentalizes the Accessible Genome

doi: 10.1038/s41588-022-01044-9

Figure Lengend Snippet: (a) Representative images of BRD2 immunofluorescence before and after acute RAD21 (500μM auxin 6 hours) or BRD4 depletion (100nM dTAG13, 6 hours) in the dual RAD21-mAID-eGFP and BRD4-Halo-dTAG mESC line. BRD2 was detected by anti-BRD2 primary antibody and goat anti-rabbit Alexa568 secondary antibody. (b) Quantification of BRD2 puncta size (left panel) and puncta mean intensity (right panel) in control (n = 1502), RAD21 depletion (n = 2314), BRD4 depletion (n = 2861) conditions, n represent the number of BRD2 puncta analyzed. (c-e ) Micro-C analysis of loops ( c ), TADs ( d) and compartments ( e ) after BRD4 depletion or dual BRD2/BRD4 depletion in the presence or absence of Cohesin. In e , saddle plots of compartmental interactions are shown. (f-g ) Micro-C analysis of active compartmental interactions (A-A interactions) ( f ) and histogram of eigenvalues from the Pearson’s correlation matrix ( g ) for BRD4 depletion alone or dual BRD4/BRD2 depletion. (h-i ) RNA-seq analysis of differentially expressed genes (DEGs) after BRD4 (h) or BRD2 (i) depletion. Volcano plot of significantly (p<0.05) expressed genes after 6 hours depletion of BRD4 or BRD2 are shown. Red dots indicate significant expressed genes with p<0.01. False positive rate adjusted p value in the -log10 form are shown in the y axis. We detected 133 DEGs for BRD2 depletion and 1579 genes for BRD4 depletion. (j) Cumulative distribution function of differentially expressed genes (DEGs) after BRD2 depletion over control relative to compartmental switches. A-A indicates no switch within the A compartment. A-I/B, switches from A to intermediate (I) or B compartment. B-I/A, switches from B to I or A compartment. The overlapping curves suggest no obvious association of DEGs with compartmental changes. (k) Profiling of chromatin accessibility by ATAC-seq before and after acute BRD2 depletion (6 hours) over DEGs (upregulated and downregulated in Fig .(i) ).

Article Snippet: The dTAG constructs was derived from pCRIS-PITChv2-dTAG-Puro (BRD4) (Addgene, 91796) and donor constructs for Brd2/3/4 were constructed by PCR amplification and Gibson assembly.

Techniques: Immunofluorescence, Control, RNA Sequencing

Journal: Nature genetics

Article Title: BRD2 Compartmentalizes the Accessible Genome

doi: 10.1038/s41588-022-01044-9

Figure Lengend Snippet: (a) Representative chromatin conformation snapshots from polymer simulation incorporating both loop extrusion and scaffold protein-mediated interactions. Simulation snapshots represent wild type (WT, incorporating Cohesin, CTCF and scaffold protein), Cohesin depletion (−), scaffold depletion (−) and dual Cohesin and scaffold depletion. See details in the in the section. (b) Polymer simulation integrating both loop extrusion and scaffold protein-mediated dynamic protein-chromatin and protein-protein interactions reproduces enhanced clustering of ACDs upon Cohesin depletion. The pair auto-correlation function g ( r ) for active chromatin beads from all recorded polymer conformations were plotted for WT and Cohesin depletion in which the scaffold protein is present or depleted (−). Removing the scaffold protein decreases the g ( r ) as observed in and . (c) The cumulative distribution function (CDF) value as a function of the 3D loci pair distance from the center of neighboring ATAC-rich segments were extracted in WT (blue curve) and Cohesin depletion (−) plus scaffold protein conditions in which both loop extrusion and scaffold protein dynamic interaction are incorporated in the revised polymer model. (d) Polymer model predictions of the segment volume of ATAC-rich segments under WT and Cohesin depletion (−) plus scaffold protein conditions. The new polymer model recapitulates experimental observations of the volume changes in - . (e) Average contact probability between ATAC-rich regions and ATAC-poor regions for WT, scaffold protein depletion, WT with protein-protein interaction reduced from 2.5k B T to 1.0k B T (non-specific) and WT with protein-protein interaction depleted (0k B T) calculated from simulations. Error bar indicates the range of average contact probability from two ATAC-rich regions from both sides with the ATAC-poor regions in the middle. k B , Boltzmann constant; T, temperature. (f ) Schematic of putative chromatin configurational changes under WT (left), Cohesin depletion (middle) or dual Cohesin/BRD2 depletion (right) conditions. ATAC-rich and ATAC-poor segments are shown in pink and cyan color, respectively. Although Cohesin loss eliminates Cohesin-dependent loops, BRD2 (and others) orchestrates extensive protein-protein and protein-chromatin interactions that lead to the enhanced chromatin interactions between ATAC-rich segments and largely unchanged 3D volume of individual ATAC-rich segments. Further BRD2 depletion mitigates interactions between active chromatin segments and decompacts individual ATAC-rich segments. (g ) Summary model. Cohesin is the master regulator of chromatin loops. BRD4 could associate with NIPBL to enhance chromatin loop formation . Both BRD4 and Cohesin could suppress BRD2 binding to chromatin, which in turn antagonize chromatin compartmentalization in euchromatin.

Article Snippet: The dTAG constructs was derived from pCRIS-PITChv2-dTAG-Puro (BRD4) (Addgene, 91796) and donor constructs for Brd2/3/4 were constructed by PCR amplification and Gibson assembly.

Techniques: Polymer, Protein-Protein interactions, Binding Assay

Journal: Cell Death & Disease

Article Title: Glutathione peroxidase 1 deficiency attenuates concanavalin A-induced hepatic injury by modulation of T-cell activation

doi: 10.1038/cddis.2016.95

Figure Lengend Snippet: Gpx1 deficiency significantly reduces cytokine production and inhibits its signaling pathway in the liver of mice. ( a ) Cytokine assay of IL-2, IFN- γ and TNF- α in the liver of WT and Gpx1 KO mice with Con A administration or without. n =8 per group; means±S.E.M., * P <0.05, WT mice without Con A administration versus WT mice with Con A administration, # P <0.05, WT mice with Con A administration versus Gpx1 KO mice with Con A administration. ( b ) Immunoblots of STAT1, JAK3 and JNK phosphorylation in the liver of WT and Gpx1 KO mice with Con A administration or without

Article Snippet: Liver and spleen tissues homogenized with protein extraction solution (PRO-PREP, iNtRON Biotechnology, Seoul, Korea) and measured the quantity of IL-2, IFN- γ and TNF- α in total proteins (1 mg) using mouse cytokine assay kit (R&D Systems, Minneapolis, MN, USA).

Techniques: Cytokine Assay, Western Blot, Phospho-proteomics

Journal: Cell Death & Disease

Article Title: Glutathione peroxidase 1 deficiency attenuates concanavalin A-induced hepatic injury by modulation of T-cell activation

doi: 10.1038/cddis.2016.95

Figure Lengend Snippet: Gpx1 deficiency reduced Con A-induced CD4+ T cells, Th1 cytokines and T-cell responses in the spleen. ( a ) Immunohistochemistry of CD4+ T cells in the spleen of WT and Gpx1 KO mice with Con A administration or without. ( b ) Cytokine assay of IL-2, IFN- γ and TNF- α in the spleen of WT and Gpx1 KO mice with Con A administration or without. n =8 per group; means±S.E.M., * P <0.05, WT mice without Con A administration versus WT mice with Con A administration, # P <0.05, WT mice with Con A administration versus Gpx1 KO mice with Con A administration. ( c ) Immunoblots of PLC γ and I κ B phosphorylation in the liver of WT and Gpx1 KO mice with Con A administration or without

Article Snippet: Liver and spleen tissues homogenized with protein extraction solution (PRO-PREP, iNtRON Biotechnology, Seoul, Korea) and measured the quantity of IL-2, IFN- γ and TNF- α in total proteins (1 mg) using mouse cytokine assay kit (R&D Systems, Minneapolis, MN, USA).

Techniques: Immunohistochemistry, Cytokine Assay, Western Blot, Phospho-proteomics

Journal: Cell Death & Disease

Article Title: Glutathione peroxidase 1 deficiency attenuates concanavalin A-induced hepatic injury by modulation of T-cell activation

doi: 10.1038/cddis.2016.95

Figure Lengend Snippet: Inhibition of Gpx activity induces T-cell hyporesponsiveness. ( a ) The splenocytes isolated from spleen of WT mice with NAC (5 mM, pharmacological antioxidant) or without, and pretreated MS (0.4 mM) for 48 h, then treated Con A (5 μ g/ml) for 8 h. Then, we performed cytokine assay. ( b ) Con A-induced mRNA expressions of Th1 cytokines such as IL-2, IFN- γ and TNF- α in MS (0.4 mM, Gpx inhibitor)-pretreated Jurkat T cell with NAC (5 mM, pharmacological antioxidant) or without. Values are expressed as the mean±S.E.M. of three different experiments conducted in triplicates. * P <0.05, control versus Con A, ## P <0.05, Con A versus pretreatment of MS then Con A, # P <0.05, pretreatment of MS then Con A versus pretreatment of MS in the presence of NAC then Con A. ( c ) BrdU incorporation assay of Con A-induced cell proliferation in MS (0.4 mM, Gpx inhibitor)-pretreated Jurkat T cell with NAC (5 mM, pharmacological antioxidant) or without using the Cell Signaling BrdU Cell Proliferation Assay Kit. Values are expressed as the mean±S.E.M. of three different experiments conducted in triplicates. * P <0.05, control versus Con A, ## P <0.05, Con A versus pretreatment of MS then Con A, # P <0.05, pretreatment of MS then Con A versus pretreatment of MS in the presence of NAC then Con A. ( d ) Immunoblots of PLC γ and I κ B phosphorylation in Con A-induced cell proliferation in MS (0.4 mM, Gpx inhibitor)-pretreated Jurkat T cell with NAC (5 mM, pharmacological antioxidant) or without

Article Snippet: Liver and spleen tissues homogenized with protein extraction solution (PRO-PREP, iNtRON Biotechnology, Seoul, Korea) and measured the quantity of IL-2, IFN- γ and TNF- α in total proteins (1 mg) using mouse cytokine assay kit (R&D Systems, Minneapolis, MN, USA).

Techniques: Inhibition, Activity Assay, Isolation, Cytokine Assay, Control, BrdU Incorporation Assay, BrdU Cell Proliferation Assay, Western Blot, Phospho-proteomics