Journal: Redox biology

Article Title: Biasing the native α-synuclein conformational ensemble towards compact states abolishes aggregation and neurotoxicity.

doi: 10.1016/j.redox.2019.101135

Figure Lengend Snippet: Fig. 1. Primary sequence and three-dimen- sional structure of wt α-syn and its double cysteine mutant α-synCC. (A) Primary se- quence alignment of wt α-syn and α-synCC. The amino acid sequences of wt α-syn (UniProt ID: P37840) and α-synCC are shown. The three distinct regions of α-syn are indicated. (B) Structural models of wt α-syn (left panel) and α-synCC (right panel) showing the NAC domain in purple. The models were generated in PyMol using the structure of pathogenic fibril of full- length human α-syn (PDB: 2N0A). Cys71 and Cys92 residues in α-synCC are shown in green.

Article Snippet: Cells were blocked with 3.5% bovine serum albumin for 2 h at RT, and incubated with primary antibody for α-syn (α-syn C-20, 1:1000, Santa Cruz) overnight at 4 °C.

Techniques: Sequencing, Mutagenesis, Generated

Journal: Redox biology

Article Title: Biasing the native α-synuclein conformational ensemble towards compact states abolishes aggregation and neurotoxicity.

doi: 10.1016/j.redox.2019.101135

Figure Lengend Snippet: Fig. 2. Sequence analysis of wt α-syn and α-synCC. (A) Aggregation pro- pensity analysis using different sequential predictors: AGGRESCAN, Zyggregator and FoldAmyloid. (B) Hydrophobicity plots. Residues exhibiting values above 0 are considered to be hydrophobic. (C) Disorder prediction with IUPred2. Residues exhibiting values above 0.5 are considered as disordered. In all plots, wt α-syn (black) and α-synCC (red).

Article Snippet: Cells were blocked with 3.5% bovine serum albumin for 2 h at RT, and incubated with primary antibody for α-syn (α-syn C-20, 1:1000, Santa Cruz) overnight at 4 °C.

Techniques: Sequencing

Journal: Redox biology

Article Title: Biasing the native α-synuclein conformational ensemble towards compact states abolishes aggregation and neurotoxicity.

doi: 10.1016/j.redox.2019.101135

Figure Lengend Snippet: Fig. 3. α-synCC disulfide formation induces compactness. (A) RP-HPLC elution profile of α-synCC before (black) and after reduction with DTT (red). (B-D) ESI- MS analysis showing the conformational ensembles of wt α-syn and α-synCC. ESI-MS spectra and Gaussian fits of the charge-state distributions obtained using 4 conformers (orange - compact conformer; green - compact intermediate state; cyan - extended intermediate state; magenta - fully extended conformer) for wt α-syn (B), oxidized α-synCC (C) and reduced α-synCC (D) are shown. The most intense peak of each ESI-MS spectra is labelled by the corresponding charge state. Inserts report the mass-deconvolution spectrum. Each Gaussian component is labelled by its relative amount in percentage. (E) 2D-TOCSY 1H–1H NMR-spectrum of wt α-syn (black) and α-synCC (red) dissolved in 20 mM HEPES, pH 7.4 at 25 °C.

Article Snippet: Cells were blocked with 3.5% bovine serum albumin for 2 h at RT, and incubated with primary antibody for α-syn (α-syn C-20, 1:1000, Santa Cruz) overnight at 4 °C.

Techniques:

Journal: Redox biology

Article Title: Biasing the native α-synuclein conformational ensemble towards compact states abolishes aggregation and neurotoxicity.

doi: 10.1016/j.redox.2019.101135

Figure Lengend Snippet: Fig. 4. Characterization of soluble wt α-syn and α-synCC. (A) Far-UV CD spectra of wt α-syn (dashed line) and its double cysteine mutant (full line). Soluble proteins at 15 µM were measured in buffer A (black), B (blue) and C (green). (B) Bis-ANS fluorescence spectra of 10 µM bis-ANS bound to 15 µM soluble wt α-syn and α-synCC in buffer A, B and C.

Article Snippet: Cells were blocked with 3.5% bovine serum albumin for 2 h at RT, and incubated with primary antibody for α-syn (α-syn C-20, 1:1000, Santa Cruz) overnight at 4 °C.

Techniques: Circular Dichroism, Mutagenesis

Journal: Redox biology

Article Title: Biasing the native α-synuclein conformational ensemble towards compact states abolishes aggregation and neurotoxicity.

doi: 10.1016/j.redox.2019.101135

Figure Lengend Snippet: Fig. 5. Aggregation kinetics of wt α-syn and α-synCC in three different buffers. The aggregation kinetics were monitored by following the change in relative Th-T fluorescence signal during 15 day-incubation of 60 µM protein in buffers A (black), B (blue) and C (green).

Article Snippet: Cells were blocked with 3.5% bovine serum albumin for 2 h at RT, and incubated with primary antibody for α-syn (α-syn C-20, 1:1000, Santa Cruz) overnight at 4 °C.

Techniques: Incubation

Journal: Redox biology

Article Title: Biasing the native α-synuclein conformational ensemble towards compact states abolishes aggregation and neurotoxicity.

doi: 10.1016/j.redox.2019.101135

Figure Lengend Snippet: Fig. 6. Characterization of wt α-syn and α-synCC aggregates formed after 5 day-incubation in buffer A, B and C. (A) Static light scattering of 10 µM wt α-syn (dashed line) and α-synCC aggregates (full line) in different buffers: A (black), B (blue) and C (green). (B) Fluorescence emission spectra of Th-T upon incubation with 10 µM wt α-syn and α-synCC fibrils formed in different buffers. (C) CR normalized absorbance spectra in the presence of 10 µM α-syn aggregates. Free CR absorbance spectrum is represented in grey. (D) TEM micrographs of negatively stained aggregates formed by wt α-syn (upper panel) and α-synCC (lower panel) in the three different conditions. Scale bars represent 200 nm. Images were taken at a 10,000× magnification. (E) PK degradation patterns of soluble wt α-syn and α-synCC (panels without frames), together with the digestion pattern of the aggregated forms (framed panels), monitored over time on Coomassie-stained SDS-PAGE. The time of digestion (in min) is indicated on the top of each panel. (F) The secondary structure of wt α-syn and α-synCC aggregates formed after 5 days. ATR-FTIR absorbance spectra in the amide I region was acquired and the fitted individual bands after Gaussian deconvolution are indicated.

Article Snippet: Cells were blocked with 3.5% bovine serum albumin for 2 h at RT, and incubated with primary antibody for α-syn (α-syn C-20, 1:1000, Santa Cruz) overnight at 4 °C.

Techniques: Incubation, Fluorescence, Staining, SDS Page

Journal: Redox biology

Article Title: Biasing the native α-synuclein conformational ensemble towards compact states abolishes aggregation and neurotoxicity.

doi: 10.1016/j.redox.2019.101135

Figure Lengend Snippet: Fig. 7. Neuroglioma cells incubated with wt α-syn or α-synCC aggregates generated in the different buffers. Cells incubated with wt α-syn produced in buffer A (A), buffer B (B) and buffer C (C). Cells incubated with α-synCC in buffer A (D), buffer B (E) and buffer C (F). Scale bar: 20 µm.

Article Snippet: Cells were blocked with 3.5% bovine serum albumin for 2 h at RT, and incubated with primary antibody for α-syn (α-syn C-20, 1:1000, Santa Cruz) overnight at 4 °C.

Techniques: Incubation, Generated, Produced

Journal: Redox biology

Article Title: Biasing the native α-synuclein conformational ensemble towards compact states abolishes aggregation and neurotoxicity.

doi: 10.1016/j.redox.2019.101135

Figure Lengend Snippet: Fig. 8. wt α-syn aggregation in the presence of α-synCC. (A) Th-T time course of fibrillization of wt α-syn in buffer B, in the absence (black line) and in the presence of increasing concentrations of soluble α-synCC (lines coloured in a gradient of blue). (B) The first 800 min of the reaction showing the aggregation lag phase. (C) Bar diagram representation of the lag time of the aggregation kinetics in the presence of increasing α-synCC concentration. Error bars indicate ± SE (n = 3). (D) TEM micrographs of aggregates generated at the final point of the monitored aggregation kinetics. Scale bars correspond to 200 nm. Images were taken at a 10,000x magnification.

Article Snippet: Cells were blocked with 3.5% bovine serum albumin for 2 h at RT, and incubated with primary antibody for α-syn (α-syn C-20, 1:1000, Santa Cruz) overnight at 4 °C.

Techniques: Concentration Assay, Generated

Journal: Redox biology

Article Title: Biasing the native α-synuclein conformational ensemble towards compact states abolishes aggregation and neurotoxicity.

doi: 10.1016/j.redox.2019.101135

Figure Lengend Snippet: Fig. 9. α-syn aggregation in the presence of lipids. (A) Change in Th-T fluorescence intensity when 70 μM wt α-syn (dots) and α-synCC (triangles) were incubated in the absence (black; upper panel) and in the presence of 70 μM DOPC (grey; middle panel) or DMPS (dark blue; lower panel) in buffer B and 37 °C. (B) TEM micrographs of fibrils generated at the final point of the aggregation kinetics. Scale bars correspond to 200 or 500 nm. Images were taken at a 10,000× magnifi- cation, except wt α-syn (DOPC) and α-synCC (DOPC) images that were taken at 8000× and 6000×, respectively.

Article Snippet: Cells were blocked with 3.5% bovine serum albumin for 2 h at RT, and incubated with primary antibody for α-syn (α-syn C-20, 1:1000, Santa Cruz) overnight at 4 °C.

Techniques: Incubation, Generated

Journal: Redox biology

Article Title: Biasing the native α-synuclein conformational ensemble towards compact states abolishes aggregation and neurotoxicity.

doi: 10.1016/j.redox.2019.101135

Figure Lengend Snippet: Fig. 10. Evaluation of wt α-syn and α-synCC oligomers conformation and toxicity. (A) Cell viability of SH-SY5Y neuroblastoma cells after 72 h-incubation in the presence of different concentrations of soluble (S) and oligomeric (O) forms of wt α-syn and α-synCC. Error bars indicate ± SE (n = 3). (B) Electron micrographs of oligomeric assemblies of wt α-syn (dashed line) and α-synCC (full line) generated after 21 h of incubation in 20 mM HEPES, pH 7.4. The scale bars correspond to 200 nm. Images were taken at a 20,000x magnification. (C) Static light scattering of 10 µM wt α-syn (dashed line) and α-synCC (full line) oligomeric assemblies. (D) Bis-ANS fluorescence spectra of 10 µM bis-ANS bound to 15 µM wt α-syn and α-synCC oligomers in 20 mM HEPES, pH 7.4. (E) Secondary structure of wt α-syn (left panel) and α-synCC oligomers (right panel). ATR-FTIR absorbance spectra in the amide I region was acquired and the fitted individual bands after Gaussian de- convolution are indicated.

Article Snippet: Cells were blocked with 3.5% bovine serum albumin for 2 h at RT, and incubated with primary antibody for α-syn (α-syn C-20, 1:1000, Santa Cruz) overnight at 4 °C.

Techniques: Incubation, Generated

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: (a) Heatmap of EZH2 ChIP-seq signal intensity in CRPC 16DCRPC and 42DENZR cell lines (left), with overlaid H3K27Ac and H3K27Me3 histone mark ChIP-seq (right). Each horizontal line represents a 6-kb locus. (b) Representative ChIP-seq tracks surrounding the WNT5A locus in 16DCRPC and 42ENZR cells. Regions of EZH2 co-occupancy with the active H3K27Ac histone mark are highlighted. (c) Relative expression of genes bound by EZH2 alone (EZH2-none) or co-operatively with H3K27Me3 (EZH2-me) and H3K27Ac (EZH2-ac) histone marks in 42DENZR and 42FENZR cell lines. Box plot shows mean and interquartile range. (d) Heatmap of H3K27Me3 and K3K27Ac ChIP-seq signal intensity surrounding AR:EZH2 co-occupied regions in 42DENZR cells. (e) Heatmap indicating AR and EZH2 ChIP-seq signal intensity at AR:EZH2 co-occupied sites (n = 2155) in 42DENZR cells, and EZH2 signal intensity at the corresponding sites in AR-negative cell lines: NCI-H660, DU145 (GEO: GSE135623), and PC-3 (GEO: GSE123204). The shade of green (AR) or blue (EZH2) reflects binding intensity. Each horizontal line represents a 6-kb locus.

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: ChIP-sequencing, Expressing, Binding Assay

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: (a) EZH2 was immunoprecipitated in 42ENZR cells, trypsin digested, and analyzed by mass spectrometry. Peptides covering 36% of EZH2 were recovered and analyzed for post-translational modifications. (n = 4 independent replicates). (b) Expression of total and phosphorylated (T350, S21, and T311 residues) EZH2 in the indicated cell lines. Protein abundance was assessed by densitometry and is reported relative to total EZH2. (c) IHC staining of pEZH2-S21 and pEZH2-T350 in serial sections from representative CRPC (n = 39) and NEPC (n = 26) patient tumours (Scale bar, 100 μm). Staining area and intensity was quantified and reported (mean ± SD; two-tailed unpaired t-test). (d) Expression of genes positively regulated by EZH2 when phosphorylated at S21 [defined by Xu et al.] in the indicated cell lines and patient tumours from the Beltran 2016 cohort. Statistical analysis was performed using a two-tailed unpaired t-test. Box plots show mean and interquartile range. ns, not significant. (e) qRT-PCR of NE lineage markers in CRPCcrEZH2 cells expressing myc-tagged EZH2S21A or EZH2S21D mutants, reported relative to empty vector transfected cells. (mean ± SD; two-tailed unpaired t-test, n = 3). Immunoblotting confirmed transgene expression. (f) Proliferation of parental 16DCRPC (control) and CRPCcrEZH2 cells stably expressing EZH2T350A and EZH2T350D phospho-mutants assessed by IncuCyte (mean ± SD, n = 3 replicates). Immunoblotting confirmed transgene expression. (g) qRT-PCR of plasticity and NE markers in VCaP and C4–2 cell lines co-transfected with EZH2 siRNA and siRNA-resistant myc-tagged EZH2WT, EZH2T350A, or EZH2T350D plasmid following treatment with ENZ (10 μM) for 7 days (mean ± SD; two-tailed unpaired t-test, n = 3).

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: Immunoprecipitation, Mass Spectrometry, Expressing, Immunohistochemistry, Staining, Two Tailed Test, Quantitative RT-PCR, Plasmid Preparation, Transfection, Western Blot, Stable Transfection

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: a, Abundance of AR, FOXA1, SUZ12 and EED peptides detected using RIME with AR antibodies as bait. Each dot represents an independent replicate, with a solid line denoting the mean. b, SUZ12 immunoprecipitation (IP) followed by immunoblotting for AR and PRC2 subunits. The relative abundance of AR was normalized to SUZ12 pulldown. c, AR–EZH2 PLA and quantification of nuclear PLA signals (red dots) from a single plane (mean ±s.d.; P < 0.0001, two-tailed unpaired t-test; n = 3). Each dot represents the number of PLA signals in a single nucleus. Scale bar, 10 μm. d, Frequency of AR-bound genes with EZH2, SUZ12 and/or EED co-occupancy based on ChIP-seq peak annotation (±50 kb from the nearest TSS) in 42DENZR cells. e, Overlap of genomic regions co-occupied by AR and EZH2 ChIP-seq peaks (AR–EZH2 complex) with ChIP-seq peaks for the H3K27Me3 and H3K27Ac in 42DENZR cells. f, Heat map of AR and EZH2 ChIP-seq signal intensity in 16DCRPC and 42DENZR cells, with corresponding ATAC-seq peak intensity. g, Overlap of AR and EZH2 ChIP-seq peaks in 16DCRPC and 42DENZR cell lines. h, Overlap of AR and EZH2 ChIP-seq peaks in the Ptenf/f;Rb1f/f (DKO) GEMM. i, Enriched reactome pathways with genes co-occupied by AR–EZH2 in 42DENZR cells and the Ptenf/f/Rb1f/f GEMM. The size of each circular data point reflects the degree to which genes in the pathway are enriched based on RNA-seq from 42DENZR compared with 16DCRPC cells. NS, not significant. j, Expression of AR–EZH2 co-bound genes in matched prostate tumours (P1–P3) pre- and post-ENZ therapy (n = 3) from the DARANA trial. Box plot shows mean and interquartile range. Statistical analysis was performed using a paired t-test. k, Venn diagram of overlap in genes downregulated (log2FC < 1) in 42DENZR cells following depletion of AR using CRISPR (crAR) or EZH2 inhibition (10 μm GSK126; 96 h). The heat map depicts relative expression of select AR–EZH2 co-bound genes, reported relative to parental cells. l, Sequential ChIP (Re-ChIP) for selected binding sites in 42DENZR cells treated with vehicle or EZH2 inhibitor (10 μm GSK126, 96 h). Cells were first analysed by chromatin immunoprecipitation with AR antibody and then immunoprecipitated again with an AR or EZH2 antibody, as indicated. Results are reported relative to IgG control (mean ± s.d., n = 2).

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: Immunoprecipitation, Western Blot, Two Tailed Test, ChIP-sequencing, RNA Sequencing Assay, Expressing, CRISPR, Inhibition, Binding Assay, Chromatin Immunoprecipitation

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: a, Expression of plasticity and neuroendocrine markers by real-time PCR (rtPCR) and Western blot in 16DCRPC cells with CRISPR-mediated EZH2 knockout (16DCRPC crEZH2) following 7 d ENZ treatment. Cells transfected with a non-silencing scrambled guide RNA (crSCR) served as a control. Data are reported relative to non-transfected cells (mean ± s.d., n = 3). Two-tailed unpaired t-test. b, Tumour growth velocity of CRPC cells with CRISPR-mediated EZH2 knockout transplanted subcutaneously into nude mice, followed by treatment with vehicle (veh) or ENZ (n = 5 mice per group). Box plots show mean and interquartile range. Mann–Whitney test. c, Gene expression analysis (by rtPCR) in 16DCRPC control and crEZH2 xenograft tumours at the experimental end point. Data are reported relative to vehicle-treated mice (mean ± s.d.; *P = 0.05, two-tailed unpaired t-test; n = 3 mice per treatment group). d, Strategy used to establish the 16Dreporter cell line carrying GFP and mCherry fluorescent reporters in the endogenous OCT4 and ASCL1 loci, respectively. Fluorescence-activated cell sorting (FACS) plot shows gating used to isolate the individual cell populations. HL, left homology arm; HR, right homology arm. e, Immunofluorescence images for OCT4-GFP (green) and ASCL1-mCherry (red) in CRPCreporter cells at the indicated time points after ENZ treatment. Single cells were tracked and are denoted with arrows. Scale bar, 100 μm. f, Fold change in transcript abundance of genes unique and common to the OCT4+, ASCL1+ and hybrid (OCT4+ASCL1+) FACS-isolated CRPCreporter cell populations relative to the negative population (log2FC cut-off of 1.5), by RNA-seq. g, MSigDB pathways enriched for common genes (n = 468) upregulated (defined as log2FC > 1.5) across OCT4+, ASCL1+ and hybrid (OCT4+ASCL1+) CRPCreporter populations relative to the negative population. Statistical analysis was performed using a hypergeometric test. h, EZH2 activity score, calculated on the basis of z-score-transformed expression of genes in the ‘Kondo EZH2 targets’ signature from MSigDB, in negative, OCT4+, ASCL1+ and hybrid (OCT4+ASCL1+) CRPCreporter FACS-isolated cell populations. i, Quantification of GFP+ and ASCL1+ fluorescent CRPCreporter cells following treatment with ENZ (10 μM) alone or in combination with EZH2 inhibitor (10 μM GSK126) using the IncuCyte fluorescent object counting algorithm (mean ± s.d., n = 2). Representative images at 8 d after treatment are shown. Scale bar, 50 μm.

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot, CRISPR, Knock-Out, Transfection, Two Tailed Test, MANN-WHITNEY, Fluorescence, FACS, Immunofluorescence, Isolation, RNA Sequencing Assay, Activity Assay, Transformation Assay

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: a, Heat map of AR, EZH2 and pEZH2-T350 ChIP-seq binding intensity in 42DENZR cells. Each horizontal line represents a 6-kb locus. b, Frequency of AR ChIP-seq peaks overlapping with EZH2 and pEZH2-T350 ChIP-seq peaks in 42DENZR cells. c, Distribution of AR–EZH2 and AR–pEZH2 co-bound peaks in relation to the TSS. Peaks were mapped into 5-kb bins. d, PLA analysis of the interaction between AR and pEZH2-T350, and quantification of nuclear PLA signals (red dots) from a single plane (mean ± s.d.; P = 3.8 × 10−10, two-tailed unpaired t-test; n = 3). Each dot represents the number of PLA signals in a single nucleus. Scale bar, 10 μm. e, Overlap of genes co-bound to AR–EZH2 occupied by SUZ12- and/or EED, based on ChIP-seq peak annotation in 42DENZR cells. Gene annotation was restricted to ±50 kb from TSS. f, Expression of genes with promoter-bound (defined as ±3 kb from TSS) AR alone or co-occupancy with EZH2 or pEZH2-T350 in 42DENZR and 42FENZR cell lines. Data are mean expression ± s.d., with significance assessed using a two-tailed unpaired t-test. g, Expression of AR–pEZH2 co-bound genes in matched individual patient tumours pre- and post-ENZ therapy from the DARANA trial (n = 3). Box plots show mean and interquartile range. Statistical analysis was performed using a paired t-test. h, Gene ontology signatures from MSigDB enriched for AR–EZH2 and AR–pEZH2 co-bound genes in 42DENZR cells. Statistical analysis was performed using a hypergeometric test. i, Immunohistochemical staining for AR, pEZH2-T350 and SYP (neuroendocrine marker) in serial sections from non-treated (naive) and neoadjuvant ADT/TAX-treated (4.5 months) prostate tumours from the CALGB 90203 clinical trial. Treated tumours were binned on the basis of pEZH2-T350 staining intensity, and matched NanoString-based sequencing was used to assess the expression of plasticity factors in pEZH2-low (n = 8) and pEZH2-high (n = 4) tumours. Box plots show mean and interquartile range of z-score-transformed expression values with significance assessed using a two-tailed unpaired t-test. Scale bar, 100 μm.

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: ChIP-sequencing, Binding Assay, Two Tailed Test, Expressing, Immunohistochemical staining, Staining, Marker, Sequencing, Transformation Assay

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: a, Immunoblot of total and phosphorylated EZH2 and CDK1 in the indicated prostate cancer cell lines. HPCS, high-plasticity cell state. b, Immunoblot of EZH2 and pEZH2-T350 following CDK1 inhibition (5 μM RO-3306, 6 h). c, Immunohistochemical staining for pEZH2-T350 and pCDK1-T161 in serial sections from treatment-naive (N, n = 30), CRPC (CR, n = 40) and NEPC (NE, n = 26) clinical samples. Scale bar, 100 μm. Staining intensity was quantified (mean ± s.d.; two-tailed unpaired t-test). d, SUZ12 and EED peptides detected by RIME using EZH2 and pEZH2-T350 antibodies as bait in 42ENZR cells. Each dot represents an independent replicate, with a solid line denoting mean. Significance was defined as ≥4 peptides. e, Myc-tagged wild-type EZH2 (EZH2WT) and T350 phospho-mimicking (EZH2T350D) and phospho-dead (EZH2T350A) mutants were transiently transfected into 16DCRPC cells with endogenous EZH2 deletion for 72 h. Immunoprecipitation was performed using a Myc tag antibody. f, Distribution of pEZH2-T350, SUZ12 and EED ChIP-seq peaks in relation to the nearest TSS. The density of polycomb subunits and H3K27Ac are shown surrounding the WNT5A locus. g, Proportion of EZH2 and pEZH2-T350 ChIP-seq peaks overlapping with H3K27Me3 and H3K27Ac ChIP-seq peaks in 42DENZR cells. The distribution of H3K27Ac alone and co-occupied with pEZH2-T350 (pEZH2-ac) in relation to the TSS is shown. h, Single-sample GSEA (ssGSEA) score of MSigDB pathways in CRPCcrEZH2 cells expressing EZH2T350A or EZH2T350D mutant, and adenocarcinoma (CRPC-Adeno) and NEPC (CRPC-NE) patient specimens from the Beltran 2016 cohort4. The ASC score is shown below each cell line or individual patient. i, rtPCR and immunoblot in 42DENZR cells with EZH2 knockdown, stably expressing siRNA-resistant Myc-tagged EZH2WT or EZH2T350A mutant for 72 h. Data are reported relative to cells transfected with empty vector (EV) (mean ± s.d.; two-tailed unpaired t-test, n = 2). j, Immunohistochemical staining for EZH2 and SYP in serial sections from CRPCcrEZH2 EZH2T350A and EZH2T350D mutant xenografts treated with vehicle or ENZ. Scale bar, 100 μm. SYP staining intensity was quantified; box plots show mean and interquartile range. k, Flow cytometry plots of CD44 and NCAM1 cell surface expression in dissociated tumour cells from EZH2T350A and EZH2T350D mutant xenografts.

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: Western Blot, Inhibition, Immunohistochemical staining, Staining, Two Tailed Test, Transfection, Immunoprecipitation, ChIP-sequencing, Expressing, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Plasmid Preparation, Flow Cytometry

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: a, PCA of global transcriptome in the indicated cell lines. 42DENZR cells with AR knockout (AR KO) and inhibited EZH2 activity (10 μM GSK126, 96 h) are shown. b, ASC and NEPC scores in patient tumours from ref. 5 (adenocarcinoma cluster 5, n = 28; AR+NE+, n = 10; AR−NE+, n = 3) and the indicated cell lines. c, GSEA signatures enriched (Fisher’s exact test, P < 0.05) in 42DENZR cells following AR knockout or EZH2 inhibition (10 μM GSK126, 96 h). d, Volcano plot of peptides detected by RIME using EZH2 antibodies as bait in 42DENZR cells treated with DMSO or EZH2 inhibitor (10 μM GSK126, 96 h). Statistical analysis was performed using a two-tailed unpaired t-test (n = 3). e, Immunoprecipitation of EZH2 in 42DENZR cells treated with 10 μM GSK126 for 96 h followed by immunoblotting. f, Immunoblot of SUZ12 in nuclear soluble and chromatin-bound fractions in 42DENZR cells treated with 10 μM GSK126 for 96 h. g, PLA analysis of AR–EZH2 in 42DENZR cells following EZH2 inhibition (10 μM GSK126, 96 h). Nuclear PLA signals from a single plane were quantified (mean ± s.d.; P = 3.1 × 10−16, two-tailed unpaired t-test; n = 3). Scale bar, 10 μm. h, Chromatin immunoprecipitation–PCR (ChIP–PCR) for AR at the AREs within the KLK3 enhancer in 42DENZR cells following treatment with EZH2 inhibitor (10 μM GSK126, 96 h). Results reported relative to IgG control (mean ± s.d.; P = 0.018, two-tailed unpaired t-test; n = 4). F, forward; R, reverse. i, rtPCR in 42DENZR cells treated with EZH2 inhibitor (10 μM GSK126 or GSK343, 96 h) or EED inhibitor (1 μM A-395, 96 h). Data reported relative to vehicle-treated cells (mean ± s.d., two-tailed unpaired t-test; n = 3). Western blot confirmed PRC2 inhibition. j, Confluency measured using IncuCyte (mean ± s.d., n = 2). At 48 h after seeding, cells were treated with EZH2 inhibitor (10 mM GSK126). k, Proliferation of 42DENZR cells treated with ENZ (10 μM) and EZH2 inhibitor (2 μM GSK126) alone or in combination, measured using IncuCyte. EZH2 inhibitor was removed (washout) at 96 h. Data plotted are mean ± s.d. (n = 3), with significance evaluated using a two-tailed unpaired t-test at the end point.

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: Knock-Out, Activity Assay, Inhibition, Two Tailed Test, Immunoprecipitation, Western Blot, Chromatin Immunoprecipitation, Reverse Transcription Polymerase Chain Reaction

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: (a) Immunoblot AR, EZH2, and H3K27Me3 (a surrogate marker of EZH2 activity) in 42DENZR cells following CRISPR-mediated AR deletion (crAR) or EZH2 inhibition (10 μM GSK126, 96 hrs). (b) Relative expression (qRT-PCR) of neuroendocrine lineage markers in 16DCRPC and C4–2 cell lines following siRNA-mediated AR silencing for 96 hours. Data are reported relative to cells transfected with a non-silencing scrambled control (mean ± SD, n = 3). A fold change >2 is considered significant. Immunoblotting confirmed AR knockdown.

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: Western Blot, Marker, Activity Assay, CRISPR, Inhibition, Expressing, Quantitative RT-PCR, Transfection

Journal: Nature cell biology

Article Title: An androgen receptor switch underlies lineage infidelity in treatment-resistant prostate cancer

doi: 10.1038/s41556-021-00743-5

Figure Lengend Snippet: (a-b) qRT-PCR in 42DENZR (a) and 42FENZR (b) cells following siRNA-mediated EZH2 silencing (siEZH2) for the indicated time, reported relative to non-transfected control cells at day 0 (mean ± SD; two-tailed unpaired t-test, n = 3). NTC, non-targeting control. (c-d) Spheroid formation and ALDH activity in 42DENZR (c) and 42FENZR (d) cells following siRNA-mediated EZH2 silencing (siEZH2; left) or treatment with increasing dose of EZH2 inhibitor (GSK126; right) for 8 days (mean ± SD; two-tailed unpaired t-test, n = 2). (e) qRT-PCR in 42DENZR cells treated with EZH2 inhibitor (10 μM GSK126) for 7 days, followed by removal (washout) for 14 days. Expression is reported relative to cells at day 0 (mean ± SD; two-tailed unpaired t-test, n = 3). Immunoblotting confirmed on-target effect.

Article Snippet: Immunohistochemical staining was performed on deparaffinized FFPE sections using a Ventana Discovery XT automated immunostainer using the following antibodies: AR (clone N-20; 1:50; Santa Cruz sc-816, lot no. G1916), pCDK1-T161 (1:400; Abcam ab47329, lot no. r3260427–5), EZH2 (Clone DR69; 1:50; Cell Signaling 5246S, lot no. 9), pEZH2-T350 (1:75; generated in this study), pEZH2-S21 (1:250; Bethyl IHC-00388, lot no. 6), and SYP (1:500; Abcam ab32127, lot no. {"type":"entrez-nucleotide","attrs":{"text":"GR223336","term_id":"238891844","term_text":"GR223336"}} GR223336 –15).

Techniques: Expressing, Activity Assay, Quantitative RT-PCR, Transfection, Two Tailed Test, Western Blot

Journal: FEBS Open Bio

Article Title: Tollip‐deficient zebrafish display no abnormalities in development, organ morphology or gene expression in response to lipopolysaccharide

doi: 10.1002/2211-5463.13423

Figure Lengend Snippet: CRISPR/Cas9‐based genome editing allows for the generation of a Tollip‐deficient zebrafish line. (A) Schemes of the zebrafish tollip transcript variants 1 and 2 (v1 and v2), based on the Ensembl database, showing exons (E), translated sequences (gray), and UTR regions (white). (B) Schematic illustration of the structure of Tollip protein isoforms with the C2 and CUE domains indicated. (C) Partial DNA sequence of the target site within exon 2 of the tollip gene in wild‐type tollip +/+ fish (left) and homozygous tollip −/− knockout fish (right). Deletion of eight nucleotides observed in the mutant line is shadowed in dark gray. There is an additional nucleotide change flanking the deletion (double peak marked R in the chromatogram, corresponding to A or G, with a predicted amino acid change D to G in the truncated protein product), indicating mosaicism of the generated line. (D) Schematic illustration of the predicted structure of Tollip protein isoforms synthesized from the mutated tollip gene. (E) Western blot of the 5 dpf protein lysates from the wild‐type ( tollip +/+ ) line and tollip −/− siblings. Top panel shows Tollip (~ 35 kDa) and a bottom panel shows α‐tubulin (~ 55 kDa) signal. (F) qPCR analysis of the expression of tollip transcripts during early zebrafish development (1–5 dpf). Bars represent the means ± SEM from 3–4 independent experiments (encompassing a pool of 10 larvae/condition). Mann–Whitney U test, * P < 0.05, ****P < 0.0001.

Article Snippet: The Cas9 RNA was synthesized from pCS2‐nCas9n (a gift from Wenbiao Chen, Addgene plasmid, #47929 [ ]) using mMESSAGE mMACHINE T7 Transcription Kit (Life Technologies, #AM1344M).

Techniques: CRISPR, Sequencing, Knock-Out, Mutagenesis, Generated, Synthesized, Western Blot, Expressing, MANN-WHITNEY

Journal: FEBS letters

Article Title: Small antisense RNA to cyclin D1 generated by pre-tRNA splicing inhibits growth of human hepatoma cells.

doi: 10.1016/j.febslet.2004.09.040

Figure Lengend Snippet: Fig. 1. Synthetic DNA sequences. Capital letters in bold indicate sequences from human tRNAtyr gene (A and B). The intron sequence of tRNAtyr (plain capital letters in B) was substituted with a 10 bp oligonucleotide (plain capital letters in A), generating two restriction sites (StuI and BamHI) to ease insertion of any antisenses, not limited to cyclin D1 as in this work. Letters in lower case represent the promoter sequence of U6 SnRNA gene (A), which enhances the transcription efficiency of subsequent pre-tRNA. The U6 SnRNA promoter and the tRNA gene were spaced by two copies of tet operator 2 sequence that serve as the binding sites for two molecules of the Tet repressor protein, so as to block the transcription of pre-tRNA from both U6 SnRNA and tRNA promoters unless with tetracycline inducement.

Article Snippet: For cyclin D1 detection, the sections were hydrated, and probed with an anti-cyclin D1 monoclonal antibody as mentioned above, and then a biotinized anti-mouse antibody and SABC complex (streptavidin–biotin-peroxidase, Boster Biotech, Wuhan, China) in sequence.

Techniques: Sequencing, Binding Assay, Blocking Assay

Journal: FEBS letters

Article Title: Small antisense RNA to cyclin D1 generated by pre-tRNA splicing inhibits growth of human hepatoma cells.

doi: 10.1016/j.febslet.2004.09.040

Figure Lengend Snippet: Fig. 2. In vitro transcription and splicing of pre-tRNA. With U6 SnRNA promoter, pre-tRNAs in pUT-tyr and pUT-ccnd1 were transcribed more efficiently than pUC-tyr control. The 20 bp anti-cy- clin D1 insert replacing the intron sequence of tRNAtyr gene was correctly spliced out (pUT-ccnd1) as well as the native intron of tRNAtyr gene (pUT-tyr).

Article Snippet: For cyclin D1 detection, the sections were hydrated, and probed with an anti-cyclin D1 monoclonal antibody as mentioned above, and then a biotinized anti-mouse antibody and SABC complex (streptavidin–biotin-peroxidase, Boster Biotech, Wuhan, China) in sequence.

Techniques: In Vitro, Control, Sequencing

Journal: FEBS letters

Article Title: Small antisense RNA to cyclin D1 generated by pre-tRNA splicing inhibits growth of human hepatoma cells.

doi: 10.1016/j.febslet.2004.09.040

Figure Lengend Snippet: Fig. 3. Western blot analysis of cyclin D1 expression. Stable blastici- din-resistant H22 cell lines were created by transfection with pTRUT- tyr or pTRUT-ccnd1. Both of the two cell lines together with un-transfected H22 control cells were induced with tetracycline for 24 h. Then, cyclin D1 expression was analyzed by Western blot. b-Actin expression was also analyzed as a control. In the figure, cyclin D1 expression was significantly reduced by cyclin D1 antisense RNA. Whereas intron of pre-tRNAtyr generated in the same way had no effect on cyclin D1 expression.

Article Snippet: For cyclin D1 detection, the sections were hydrated, and probed with an anti-cyclin D1 monoclonal antibody as mentioned above, and then a biotinized anti-mouse antibody and SABC complex (streptavidin–biotin-peroxidase, Boster Biotech, Wuhan, China) in sequence.

Techniques: Western Blot, Expressing, Transfection, Control, Generated

Journal: FEBS letters

Article Title: Small antisense RNA to cyclin D1 generated by pre-tRNA splicing inhibits growth of human hepatoma cells.

doi: 10.1016/j.febslet.2004.09.040

Figure Lengend Snippet: Fig. 6. Immunohistochemical analysis of cyclin D1 expression and apoptosis in s.c. tumor xenografts with and without plasmid pUT-ccnd1 injection. Preformed tumor xenografts were harvested at day 17 after 6 times of plasmid injection. Cyclin D1 expression (A) and apoptosis (B) were analyzed as described in Section 2. (a) s.c. tumors treated with TE only; (b) s.c. tumors treated with plasmid pUT-ccnd1.

Article Snippet: For cyclin D1 detection, the sections were hydrated, and probed with an anti-cyclin D1 monoclonal antibody as mentioned above, and then a biotinized anti-mouse antibody and SABC complex (streptavidin–biotin-peroxidase, Boster Biotech, Wuhan, China) in sequence.

Techniques: Immunohistochemical staining, Expressing, Plasmid Preparation, Injection

Journal: FEBS letters

Article Title: Small antisense RNA to cyclin D1 generated by pre-tRNA splicing inhibits growth of human hepatoma cells.

doi: 10.1016/j.febslet.2004.09.040

Figure Lengend Snippet: Fig. 5. Effect of plasmid administration on tumor growth. Four days after inoculation of H22 cells (Day 5), pUT-ccnd1 plasmid was injected into the s.c. tumor xenografts at multiple sites until day 11 or day 15. Tumor volumes were determined by bidimensional caliper measure- ments and were presented as the mean tumor volume (n ¼ 8). The bars stand for standard deviation. (*) Significant (P < 0:05) volume differ- ence between plasmid treated tumors and TE treated control tumors. ðmÞ Significant (P < 0:05) difference between short-term plasmid treated tumors (to day 11) and long-term treated tumors (to day 15). Fig. 4. Cell proliferation assay by MTT method. H22 cells were stained by MTT. Proliferation of the cells was significantly (P < 0:05) reduced by the antisense RNA. The bars represent the standard deviations calculated from three repeated experiments.

Article Snippet: For cyclin D1 detection, the sections were hydrated, and probed with an anti-cyclin D1 monoclonal antibody as mentioned above, and then a biotinized anti-mouse antibody and SABC complex (streptavidin–biotin-peroxidase, Boster Biotech, Wuhan, China) in sequence.

Techniques: Plasmid Preparation, Injection, Standard Deviation, Control, Proliferation Assay, Staining