Journal: Cell Reports

Article Title: CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci

doi: 10.1016/j.celrep.2020.108545

Figure Lengend Snippet:

Article Snippet: Mouse anti-O-GlcNAC-AF647 (Clone RL2) , NOVUS Biologicals , Cat#NB300-524AF647; RRID: AB_10001871.

Techniques: Purification, Recombinant, Phospho-proteomics, Software

Journal: bioRxiv

Article Title: ER-tethering directs TREX1 penetration of a BAF-dependent barrier at micronuclei

doi: 10.1101/2025.04.14.648782

Figure Lengend Snippet: (A) Schematics of micronuclei purification workflow. (B) Flow profiles of partially purified micronuclei from HeLa H2B-mCherry GFP-cGAS cells. (C) Volcano plots showing relative protein abundances in ruptured versus total micronuclei in HeLa H2B-mCherry GFP-cGAS cells. Displayed proteins were identified at FDR<0.01. Relative abundance is expressed as log₂-transformed ratio of the total area-under-the-curve for peptides assigned to each protein in different conditions. Total LFQ intensities (Label-Free Quantification, au) assigned to each protein is used as surrogate for signal-to-noise and accuracy of quantification. GFP-cGAS is used as a marker for ruptured micronuclei. Two independent experiments were performed. (D) Volcano plots showing relative protein abundances in ruptured versus intact micronuclei, as in (C). (E) Live cell imaging of endogenously tagged HeLa GFP 11 -BAF GFP 1-10 cells. Scale bar, 10 µm. (F) Quantification of GFP-BAF micronuclei over primary nuclei signal intensity ratio as shown in (E). Mean ± s.d. of individual cells from n = 1 experiment are shown. (G) Quantification of LEM domain proteins micronuclei over primary nuclei signal intensity ratio as shown in Figure S1E. Mean ± s.d. of n = 3 experiments are shown with greater than 100 total micronuclei quantified per experiment. P values were calculated by Student’s t -test (**P < 0.01, *P < 0.1) (H) Immunoblotting for the indicated proteins in MN sorted from HeLa H2B-mCherry GFP-cGAS cells. See also Figure S1.

Article Snippet: Using a split GFP system , GFP 1-10 (Addgene plasmid #80409) was stably expressed in HeLa iCas9 parental cells and CRISPR/Cas9-mediated HDR was performed to knock in GFP 11 -3×FLAG at the N-terminus of TREX1 ( ).

Techniques: Purification, Transformation Assay, Quantitative Proteomics, Marker, Live Cell Imaging, Western Blot

Journal: bioRxiv

Article Title: ER-tethering directs TREX1 penetration of a BAF-dependent barrier at micronuclei

doi: 10.1101/2025.04.14.648782

Figure Lengend Snippet: (A) Schematics of targeting GFP 11 to TREX1 N terminus in HeLa GFP 1-10 cells. (B) Immunoblotting of HeLa GFP 1-10 GFP 11 -TREX1 with the indicated antibodies. (C) Live-cell imaging of HeLa GFP 1-10 GFP 11 -TREX1 along with NLS-RFP. DNA was stained with Hoechst. Scale bar, 5 µm. (D) Quantification of the percentage of GFP-TREX1 positive micronuclei as shown in (C). Mean ± s.d. of n = 4 independent experiments with greater than 100 total micronuclei quantified per experiment. P value was calculated by Student’s t -test (****P < 0.0001). (E) siRNA screen quantification of percentage of GFP-TREX1 positive micronuclei to identify factors required for TREX1 recruitment to ruptured MN. Mean ± s.d.; ****P < 0.0001, ***P < 0.001, ordinary one-way ANOVA with Dunnett’s multiple comparisons test; > 3 independent experiments for each siRNA treatment. From left to right: Total MN analyzed: n = 1589, 567, 494, 612, 342, 232, 295, 273, 278, 386, 358, 694, 465, 599, 387; Ruptured MN analyzed: n = 967, 441, 385, 453, 201, 169, 216, 183, 170, 268, 247, 360, 287, 298, 229. (F) Live-cell imaging of HeLa GFP 1-10 GFP 11 -TREX1 NLS-RFP with BAF depletion by siRNA. Scale bar, 10 µm. (G) Quantification of the normalized fluorescence signal intensities of GFP-TREX1 MN/ER ratio in HeLa stably expressing GFP-TREX1 with BAF depletion by siRNA. Mean ± s.d. of n = 3 independent experiments are shown with greater than 100 total micronuclei quantified per experiment. P value was calculated by Student’s t -test (*P < 0.1). (H) Live-cell time-lapse imaging of HeLa inducible BAF KO stably expressing GFP-TREX1 after 72 hours treatment of dmso ( BANF1 WT) or doxycycline ( BANF1 KO), captured at 10-minute intervals. Time 0 min marks the micronuclear envelope rupture, indicated by the loss of NLS-RFP signal. Scale bar, 10 µm. (I) Quantification of the normalized fluorescence signal intensities of GFP-TREX1 MN/ER ratio as shown in (H). Mean ± s.e.m. ***P<0.001. Wilcoxon matched-pairs signed rank test; 3 independent experiments. BANF1 wt n = 23 ruptured MN; BANF1 KO n = 26 ruptured MN. See also Figure S2.

Article Snippet: Using a split GFP system , GFP 1-10 (Addgene plasmid #80409) was stably expressed in HeLa iCas9 parental cells and CRISPR/Cas9-mediated HDR was performed to knock in GFP 11 -3×FLAG at the N-terminus of TREX1 ( ).

Techniques: Western Blot, Live Cell Imaging, Staining, Fluorescence, Stable Transfection, Expressing, Imaging

Journal: bioRxiv

Article Title: ER-tethering directs TREX1 penetration of a BAF-dependent barrier at micronuclei

doi: 10.1101/2025.04.14.648782

Figure Lengend Snippet: (A) Live-cell time-lapse imaging of HeLa inducible BANF1 KO after 72 hours treatment of dmso ( BANF1 WT) or doxycycline ( BANF1 KO) and stained with ER tracker Green, captured at 10-minute intervals. Arrow indicated a micronucleus going through rupture. Time 0 min marks the micronuclear envelope rupture, indicated by the loss of NLS-RFP signal. Scale bar, 10 µm. (B) Quantification of the normalized fluorescence signal intensities of ER tracker as shown in (A). Mean ± s.e.m. ***P<0.001. Wilcoxon matched-pairs signed rank test; 3 independent experiments. BANF1 wt n = 34 ruptured MN; BANF1 KO n = 51 ruptured MN. (C) Schematics of BAF separation of function mutants. (D) Immunofluorescence of HeLa parental cells stably expressing EGFP-BAF-variants and NLS-RFP. Arrows marked ruptured micronuclei indicated by the loss of NLS-RFP signal. Scale bar, 10 µm.(E) Quantification of normalized EGFP-BAF intensity at ruptured micronuclei as shown in (D). Mean and SEM of n = 3 independent experiments with greater than 100 total micronuclei quantified per experiment. Ordinary one-way ANOVA with Dunnett’s multiple comparisons test (****P < 0.0001). (F) Live-cell time-lapse imaging of HeLa inducible BANF1 KO stably expressing GFP-TREX1 rescued by Myc-BAF-WT or L58R after 72 hours treatment of doxycycline ( BANF1 KO), captured at 10-minute intervals. Time 0 min marks the micronuclear envelope rupture, indicated by the loss of NLS-RFP signal. Scale bar, 10 µm. Scale bar, 10 µm. (G) Quantification of the normalized fluorescence signal intensities of GFP-TREX1 MN/ER ratio as shown in (F). Mean ± s.e.m. ****P<0.0001. Wilcoxon matched-pairs signed rank test; n = 3 independent experiments. BAF WT n = 22 ruptured MN; BAF L58R n = 29 ruptured MN. See also Figure S3.

Article Snippet: Using a split GFP system , GFP 1-10 (Addgene plasmid #80409) was stably expressed in HeLa iCas9 parental cells and CRISPR/Cas9-mediated HDR was performed to knock in GFP 11 -3×FLAG at the N-terminus of TREX1 ( ).

Techniques: Imaging, Staining, Fluorescence, Immunofluorescence, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: ER-tethering directs TREX1 penetration of a BAF-dependent barrier at micronuclei

doi: 10.1101/2025.04.14.648782

Figure Lengend Snippet: (A) Live-cell time-lapse imaging of HeLa inducible BAF KO stably expressing GFP-RPA after 72 hours treatment of dmso ( BANF1 WT) or doxycycline ( BANF1 KO), captured at 10-minute intervals. Arrow indicated a micronucleus going through rupture. Time 0 min marks the micronuclear envelope rupture, indicated by the loss of NLS-RFP signal. Scale bar, 10 µm. (B) Quantification of the fraction of RPA + ruptured micronuclei as shown in (A). Mean ± s.e.m. ****P<0.0001. Wilcoxon matched-pairs signed rank test; 3 independent experiments. BANF1 wt n = 28 ruptured MN; BANF1 KO n = 42 ruptured MN. (C) Immunofluorescence of RPA in HeLa iCas9 sgBAF expressing NLS-RFP. Arrows marked ruptured micronuclei indicated by the loss of NLS-RFP signal. Scale bar, 10 µm. (D) Quantification of normalized RPA intensity in ruptured micronuclei as in (C). (E) Immunofluorescence of RPA in HeLa TREX1 KO treated with CTRL or BAF siRNA. Arrows marked ruptured micronuclei indicated by the loss of NLS-RFP signal. Scale bar, 10 µm. (F) Quantification of normalized RPA intensity in ruptured micronuclei as in (E). (G) Immunofluorescence of native BrdU in HeLa iCas9 sgBAF expressing NLS-RFP. Arrows marked ruptured micronuclei indicated by the loss of LaminB1 signal. Scale bar, 10 µm. (H) Quantification of normalized BrdU intensity in ruptured micronuclei as in (G). (I) Immunofluorescence of γH2AX in HeLa iCas9 sgBAF expressing NLS-RFP. Arrows marked ruptured micronuclei indicated by the loss of NLS-RFP signal. Scale bar, 10 µm (J) Quantification of normalized γH2AX intensity in ruptured micronuclei as in (I). P values in (D)(F)(H)(J) were calculated by Student’s t -test (***P < 0.001; ****P < 0.0001). Mean ± s.e.m. of n = 3 independent experiments with greater than 100 total micronuclei quantified per experiment. See also Figure S4.

Article Snippet: Using a split GFP system , GFP 1-10 (Addgene plasmid #80409) was stably expressed in HeLa iCas9 parental cells and CRISPR/Cas9-mediated HDR was performed to knock in GFP 11 -3×FLAG at the N-terminus of TREX1 ( ).

Techniques: Imaging, Stable Transfection, Expressing, Immunofluorescence

Journal: bioRxiv

Article Title: ER-tethering directs TREX1 penetration of a BAF-dependent barrier at micronuclei

doi: 10.1101/2025.04.14.648782

Figure Lengend Snippet: (A) Representative DNA gel from in vitro nuclease assay. A dsDNA substrate was co-incubated with a fixed concentration of purified human TREX1 protein together with the indicated concentration of purified human BAF protein. - = no TREX1 added. (B) Representative DNA gel from in vitro nuclease assay, using purified BAF K6A mutant with deficient DNA binding instead of wild type BAF in (A). (C) Representative DNA gel from in vitro nuclease assay, using purified human cGAS instead of wild type BAF in (A). (D) Quantification of the in vitro nuclease assay in (A)(B)(C); mean ± s.d., n = 3, Wilcoxon matched-pairs signed rank test between BAF and BAF K6A (**P<0.01). (E) Schematics of human TREX1 transgenes. FL = full length, TMD = trans-membrane domain, IDR = intrinsically disordered region. (F) Immunofluorescence of RPA in HeLa TREX1 KO cells stably expressing GFP-TREX1ΔTMD as shown in (E) and treated with CTRL or BAF siRNA. Arrows marked ruptured micronuclei indicated by the loss of NLS-RFP signal. Scale bar, 10 µm. (G) Quantification of normalized RPA intensity in ruptured micronuclei as shown in (F). Mean ± s.e.m., n = 3, Student’s t -test (****P < 0.0001), each experiment with greater than 100 total micronuclei quantified. See also Figure S5.

Article Snippet: Using a split GFP system , GFP 1-10 (Addgene plasmid #80409) was stably expressed in HeLa iCas9 parental cells and CRISPR/Cas9-mediated HDR was performed to knock in GFP 11 -3×FLAG at the N-terminus of TREX1 ( ).

Techniques: In Vitro, Nuclease Assay, Incubation, Concentration Assay, Purification, Mutagenesis, Binding Assay, Membrane, Immunofluorescence, Stable Transfection, Expressing

Journal: bioRxiv

Article Title: Cytoplasmic Colocalization of Granulins and TDP-43 Prion-like Domain Involves Electrostatically Driven Coacervation Tuned by the Redox State of Cysteines

doi: 10.1101/2021.06.25.449959

Figure Lengend Snippet: Confocal microscopy images of live SH-SY5Ycells under homeostatic or stress conditions. a) Blue fluorescent protein tagged PrLD (PrLD-SBFP2) transiently expressed in SH-SY5Y cells alone and in the presence of Hilyte-532 labeled GRN-2 under non-stress conditions, and b) the same reactions under stress induced by sodium arsenite. White arrows indicate colocalized GRN-2 and PrLD puncta in the cytoplasm, while yellow arrows represent colocalization of GRN-2 in lysosomes (Scale bar represents 5 μm). Visualization of nucleus and lysosomes were done by staining with NucSpot ® Live 650 and Lysoview™ 650 respectively c) Normalized intensities of fluorescence recovery after photobleaching (FRAP) for puncta of PrLD alone or colocalized PrLD-GRN-2 in under non-stress (top) or stress (bottom) conditions. d) Whiskers plot of Manders’ tM1 calculated for the colocalization of GRN-2 with PrLD using Fiji-ImageJ software. Each data-point represents the colocalization score of an independent cell (n = 14).

Article Snippet: Cells were stained with nuclear (NucSpot ® Live 650, Biotium) or lysosomal (Lysoview™ 650, Biotium) markers prior to imaging at 40X magnification using Leica STELLARIS-DMI8 microscope.

Techniques: Confocal Microscopy, Labeling, Staining, Fluorescence, Software

Journal: Cell reports

Article Title: Molecular motor protein KIF5C mediates structural plasticity and long-term memory by constraining local translation

doi: 10.1016/j.celrep.2021.109369

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: NaCl (5M)- RNase free , Thermo Fisher scientific , AM9760G.

Techniques: Virus, Recombinant, Cell Culture, Saline, Sterility, Live Cell Imaging, Transfection, Protein Extraction, Protease Inhibitor, Labeling, SYBR Green Assay, Ointment, Adhesive, Gel Extraction, Isolation, TA Cloning, Sequencing, Plasmid Preparation, shRNA, Software, Control