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: eLife

Article Title: NAB2-STAT6 drives an EGR1-dependent neuroendocrine program in solitary fibrous tumors

doi: 10.7554/eLife.98072

Figure Lengend Snippet: ( a ) Table of the eight formalin-fixed paraffin-embedded (FFPE) samples used for RNA-seq listing the sample ID, Age at surgery, sex, diagnosis of tumor status, and site of resection. ( b ) Graphic demonstrating the workflow for FFPE RNA-seq. RNA was extracted from FFPE tumor and matching normal tissue and sequenced using exon-targeted sequencing which increases quality of FFPE RNA-seq’s. ( c ) Most abundant gene fusion present in all solitary fibrous tumors (SFTs) in FFPE RNA-seq, NAB2-STAT6 with exons 1–4 of NAB2 and exons 2–22 of STAT6. Graphic generated with Arriba, a fusion detection algorithm. ( d ) Volcano plot showing differentially expressed genes in SFTs versus normal matching tissues as determined by FFPE RNA-seq (n=8). 2429 genes were upregulated (indicated by red dots) and 3769 genes were downregulated (indicated by blue dots). Fold change >1, FDR <0.1. ( e ) Biological pathway gene ontology (GO) analysis of 2429 upregulated genes in INTS10 KO cells revealed enrichment for developmental and specifically neuronal developmental pathways. ( f ) TRANSFAC motif (transcription factor motifs at +/-1 kb from transcription start site) GO analysis of 2429 upregulated genes in INTS10 KO cells revealed enrichment for DB1, MAZ, MOVO-B, EGR1, and WT1 motifs.

Article Snippet: Antibody , STAT6 , Santa Cruz Biotechnology , Cat#sc-374021 , WB, 1:1000, IF, 1:500, ChIP.

Techniques: Formalin-fixed Paraffin-Embedded, RNA Sequencing, Biomarker Discovery, Sequencing, Generated

Journal: eLife

Article Title: NAB2-STAT6 drives an EGR1-dependent neuroendocrine program in solitary fibrous tumors

doi: 10.7554/eLife.98072

Figure Lengend Snippet: ( a ) Chromosomal rearrangements present in SFT 004 detected by Arriba. The inversion producing NAB2-STAT6 was the most abundant. ( b ) Spearman’s correlation analysis of gene expression between 26 previously published RNA-seqs of solitary fibrous tumors (SFTs) and our eight SFTs and normal matching tissues from the formalin-fixed paraffin-embedded (FFPE) RNA. ( c ) Principal component analysis (PCA) of FFPE RNA-seq datasets of SFTs and normal matching tissue. All SFTs cluster away from Normal matching tissue along the PC1 axis (27% of variance), suggesting broad and consistent transcriptome changes in SFTs. ( d ) Biological pathway gene ontology (GO) analysis of 3769 downregulated genes in SFTs cells revealed enrichment for immune and cell signaling pathways. ( e ) Dot plot of gene set enrichment analysis (GSEA) analysis shows the upregulation of neuronal signatures in SFT and downregulation of immune signatures. ( f ) TRANSFAC motif (transcription factor motifs at +/-1 kb from transcription start site) GO analysis of 3769 downregulated genes in SFTs revealed enrichment for HTF4 and MRF4, transcription factors that are usually active in mesoderm-derived tissues.

Article Snippet: Antibody , STAT6 , Santa Cruz Biotechnology , Cat#sc-374021 , WB, 1:1000, IF, 1:500, ChIP.

Techniques: Gene Expression, Formalin-fixed Paraffin-Embedded, RNA Sequencing, Protein-Protein interactions, Derivative Assay

Journal: eLife

Article Title: NAB2-STAT6 drives an EGR1-dependent neuroendocrine program in solitary fibrous tumors

doi: 10.7554/eLife.98072

Figure Lengend Snippet: ( a ) We generated a doxycycline-inducible clone that expresses NAB2-STAT6 (NAB2 exons 1–4, STAT6 exons 2–22) with a C-terminal FLAG tag. Immunoblot analysis of whole cell extracts shows strong expression of NAB2-STAT6 after 1, 2, and 3 days of doxycycline treatment using a FLAG antibody. GAPDH was used as control. ( b ) Heatmap clustering analysis of 2430 genes that are differentially expressed (fold change >1, FDR <0.1) across 1, 2, and 3 days of NAB2-STAT6 expression (Dox) as determined by 3' mRNA Quant-seq (n=4) ( c ) Volcano plot showing differentially expressed genes in cells expressing NAB2-STAT6 (Dox) for 2 days versus control cells as determined by 3' mRNA Quant-seq (n=4). 562 genes were upregulated (indicated by red dots) and 211 genes were downregulated (indicated by blue dots). Fold change >1, FDR <0.1. ( e ) Biological pathway GO analysis of 562 upregulated genes in INTS10 KO cells revealed enrichment for neuronal developmental pathways. ( e ) TRANSFAC motif (transcription factor motifs at +/-1 kb from transcription start site) gene ontology (GO) analysis of 562 upregulated genes in INTS10 KO cells revealed enrichment for EGR1 and PATZ motifs. ( f ) We plotted normalized read counts of NAB1, NAB2, EGR1, EGR2, STAT6, EGR target IGF2, and neuronal markers m (LHX2, ROBO2, and SHOX2) over 3 days of NAB2-STAT6 (Dox) expression. Targets of EGR1 were gradually upregulated; NAB1, NAB2, EGR1, IGF2, LHX2, ROBO2, and SHOX2 . Figure 2—source data 1. Original scans with captions. Figure 2—source data 2. Raw immunoblot scans for .

Article Snippet: Antibody , STAT6 , Santa Cruz Biotechnology , Cat#sc-374021 , WB, 1:1000, IF, 1:500, ChIP.

Techniques: Generated, FLAG-tag, Western Blot, Expressing, Control

Journal: eLife

Article Title: NAB2-STAT6 drives an EGR1-dependent neuroendocrine program in solitary fibrous tumors

doi: 10.7554/eLife.98072

Figure Lengend Snippet: ( a ) Principal component analysis (PCA) of 3' mRNA Quant-seq (n=4) datasets of Dox treated (1, 2, or 3 days) and control cells. The longer NAB2-STAT6 is expressed the more cells (Dox) move further along the PC1 axis (27% of variance), suggesting NAB2-STAT6 induces broad and consistent transcriptome changes in U2OS cells. ( b ) Biological pathway gene ontology (GO) analysis of 299 upregulated genes in cluster 1 of heatmap from . revealed enrichment for translation and biosynthetic pathways. ( c ) TRANSFAC motif (transcription factor motifs at +/-1 kb from transcription start site) GO analysis 299 upregulated genes in cluster 1 of heatmap from revealed enrichment for DP-1 and ZF5 motifs. ( d ) Heatmap clustering analysis of 166 genes that are differentially expressed (fold change >1, FDR <0.1) across 1, 2, and 3 days of NAB2-STAT6 expression (Dox) and solitary fibrous tumors (SFTs) as determined by 3' mRNA Quant-seq (n=4). Includes EGR1 targets like IGF2, LHX2, and ROBO2.

Article Snippet: Antibody , STAT6 , Santa Cruz Biotechnology , Cat#sc-374021 , WB, 1:1000, IF, 1:500, ChIP.

Techniques: Control, Expressing

Journal: eLife

Article Title: NAB2-STAT6 drives an EGR1-dependent neuroendocrine program in solitary fibrous tumors

doi: 10.7554/eLife.98072

Figure Lengend Snippet: ( a ) Average profiles and heatmaps of NAB2-STAT6 FLAG and EGR1 ChIP-seq and ATAC-seq in both control and 2 days NAB2-STAT6 (Dox) expressing U2OS cells at 1394 NAB2-STAT6 FLAG peaks. NAB2-STAT6 FLAG becomes significantly localized to these peaks which have significant increases in EGR1 and ATAC-seq signal. ( b ) Motif analysis of 1394 NAB2-STAT6 FLAG peaks using HOMER shows EGR1, EGR2, and WT1 as the most significantly enriched TF matrices. ( c ) Gene set enrichment analysis (GSEA) shows that genes nearest to NAB2-STAT6 FLAG peaks (n=1394) are significantly upregulated after 2 days of NAB2-STAT6 (Dox) expression in U2OS cells when compared with control cells from . ( d ) Screenshot displays two enhancers and the promoter (highlighted in yellow) of KNDC1 that gains NAB2-STAT6 FLAG localization and has increases in EGR1 localization and accessibility by ATAC-seq. ( e ) Screenshot displays an enhancer (highlighted in yellow) of IGF2 that gains NAB2-STAT6 FLAG localization and has increased EGR1 localization and accessibility by ATAC-seq.

Article Snippet: Antibody , STAT6 , Santa Cruz Biotechnology , Cat#sc-374021 , WB, 1:1000, IF, 1:500, ChIP.

Techniques: ChIP-sequencing, Control, Expressing

Journal: eLife

Article Title: NAB2-STAT6 drives an EGR1-dependent neuroendocrine program in solitary fibrous tumors

doi: 10.7554/eLife.98072

Figure Lengend Snippet: ( a ) Pie chart showing the percentage of enhancer (1 kb >from nearest TSS) and promoter (1 kb

Article Snippet: Antibody , STAT6 , Santa Cruz Biotechnology , Cat#sc-374021 , WB, 1:1000, IF, 1:500, ChIP.

Techniques: Control, Expressing

Journal: eLife

Article Title: NAB2-STAT6 drives an EGR1-dependent neuroendocrine program in solitary fibrous tumors

doi: 10.7554/eLife.98072

Figure Lengend Snippet: ( a ) The most abundant gene fusion present in the primary solitary fibrous tumor (SFT) used in by RNA-seq was NAB2-STAT6 with exons 1–6 of NAB2 and exons 17–22 of STAT6. Graphic generated with Arriba, a fusion detection algorithm. ( b ) Venn diagram of overlapping NAB2 and STAT6 peaks from ChIP-seq in control U2OS cells showing minimal overlap in physiological conditions confirming validity of dual antibody from . ( c ) Pie chart showing the percentage of enhancer (1 kb >from nearest TSS) and promoter (1 kb

Article Snippet: Antibody , STAT6 , Santa Cruz Biotechnology , Cat#sc-374021 , WB, 1:1000, IF, 1:500, ChIP.

Techniques: RNA Sequencing, Generated, ChIP-sequencing, Control

Journal: eLife

Article Title: NAB2-STAT6 drives an EGR1-dependent neuroendocrine program in solitary fibrous tumors

doi: 10.7554/eLife.98072

Figure Lengend Snippet: ( a ) Summary of the strategy to profile NAB2-STAT6 binding in a primary solitary fibrous tumor (SFT). The primary tumor was designated and single cells were isolated and fixed. Fixed cells were then used for NAB2 and STAT6 ChIP-seq. Peaks overlapping in both NAB2 and STAT6 ChIP-seq were characterized as NAB2-STAT6 peaks. ( b ) Average profiles and heatmaps of NAB2, STAT6, and RNAPII ChIP-seq in a primary SFT at 5921 NAB2 only peaks, 718 NAB2-STAT6 peaks, and 1285 STAT6 only peaks. NAB2-STAT6 peaks had significant NAB2, STAT6, and RNAPII signal. ( c ) Top 2 motifs from motif analysis of 5921 NAB2 only peaks, 718 NAB2-STAT6 peaks, and 1285 STAT6 only peaks using HOMER shows EGR1 and WT1 as the most significantly enriched TF matrices at NAB2 and NAB2-STAT6 sites and GRE and STAT3 at STAT6 sites. ( d ) GSEA shows that genes nearest to NAB2-STAT6 peaks (n=718) are significantly upregulated in SFTs when compared with matching normal tissue from . ( e ) Screenshot displays the promoter (highlighted in yellow) of KLF10 that has significant NAB2, STAT6, and RNAPII localization in SFTs and in U2OS gains NAB2-STAT6 FLAG localization and has increased EGR1 localization and accessibility by ATAC-seq. ( f ) Screenshot displays an enhancer and promoter (highlighted in yellow) of NAB2 that has significant NAB2 and STAT6 localization in SFTs and in U2OS gains NAB2-STAT6 FLAG localization and has increased EGR1 localization and accessibility by ATAC-seq.

Article Snippet: Antibody , STAT6 , Santa Cruz Biotechnology , Cat#sc-374021 , WB, 1:1000, IF, 1:500, ChIP.

Techniques: Binding Assay, Isolation, ChIP-sequencing

Journal: eLife

Article Title: NAB2-STAT6 drives an EGR1-dependent neuroendocrine program in solitary fibrous tumors

doi: 10.7554/eLife.98072

Figure Lengend Snippet: ( a ) Eluates from NAB2 and STAT6 IPs from U2OS nuclear extracts expressing NAB2-STAT6 for 1 day were subjected to MudPIT LC-MS/MS analysis for unbiased identification of the top interactors. Log2 iBAQ protein scores of STAT6 IP interactors are plotted against scores of NAB IP. NAB1 and EGR1 were the top interactors. ( b ) Control and U2OS cells expressing NAB2-STAT6 for 1 day were subcellularly fractionated into nuclear and cytoplasmic fractions. Immunoblot analysis shows that NAB2-STAT6 was only present in the nuclear fraction of Dox conditions. STAT6 was nuclear in both conditions. NAB2 and EGR1 were cytoplasmic in control conditions but became nuclear in Dox conditions. GAPDH was cytoplasmic control and Histone H3 was nuclear control. ( c ) Immunocytochemistry (ICC) of NAB2 (red), STAT6 (green), and DAPI (blue) in solitary fibrous tumor (SFT) primary cells from and U2OS control and NAB2-STAT6 (Dox) expressing for one day cells. SFT and NAB2-STAT6 expressing cells show strong nuclear staining for NAB2 and STAT6. Control U2OS cells have nuclear STAT6 and cytoplasmic NAB2 staining. ( d ) Immunocytochemistry (ICC) of NAB1 (red), FLAG (green), and DAPI (blue) in U2OS control and NAB2-STAT6 (Dox) expressing cells for 1 day. NAB2-STAT6 expressing cells show strong nuclear staining for FLAG and NAB1. Control U2OS cells have no FLAG and cytoplasmic and nuclear NAB1 staining. Figure 5—source data 1. Immunoblot scans with captions. Figure 5—source data 2. Raw immunoblot scans for .

Article Snippet: Antibody , STAT6 , Santa Cruz Biotechnology , Cat#sc-374021 , WB, 1:1000, IF, 1:500, ChIP.

Techniques: Expressing, Liquid Chromatography with Mass Spectroscopy, Control, Western Blot, Immunocytochemistry, Staining

Journal: eLife

Article Title: NAB2-STAT6 drives an EGR1-dependent neuroendocrine program in solitary fibrous tumors

doi: 10.7554/eLife.98072

Figure Lengend Snippet: ( a ) Eluates from EGR1 IPs from control U2OS and U2OS nuclear extracts expressing NAB2-STAT6 for 1 day were subjected to MudPIT LC-MS/MS analysis for unbiased identification of the top interactors. Log2 iBAQ protein scores of control IP interactors are plotted against scores of NAB2-STAT6 expressing IP. NAB2 and STAT6 were only pulled down in NAB2-STAT6 expressing cells. ( b ) We generated a clone that constitutively expressed NAB2-STAT6 (NAB2 exons 1–4, STAT6 exons 2–22) with a C-terminal FLAG tag in HEK293T. Immunoblot analysis of whole cell extracts shows strong expression of NAB2-STAT6. GAPDH was used as control. ( C ) Eluates from FLAG IPs from U2OS nuclear extracts expressing NAB2-STAT6 for 1 day and HEK293T clone #1 constitutively expressing NAB2-STAT6 were subjected to MudPIT LC-MS/MS analysis for unbiased identification of the top interactors. Log2 iBAQ protein scores of U2OS IP interactors are plotted against scores of HEK293T IP. NAB1 was the top interactor. ( d ) Peptide counts pulled down from IP-MS. ( e ) Map showing the position of peptides pulled down by U2OS NAB2-STAT6 FLAG IP on NAB2. Peptides were pulled down that are only present in the WT NAB2, not NAB2-STAT6. ( f ) Map showing the position of peptides pulled down by HEK293T NAB2-STAT6 FLAG IP on NAB2. Peptides were pulled down that are only present in the WT NAB2, not NAB2-STAT6. ( g ) Immunocytochemistry (ICC) of NAB2 (red), STAT6 (green), and DAPI (blue) in WT U2OS cells. NAB2 was primarily cytoplasmic and STAT6 primarily nuclear. ( h ) Immunocytochemistry (ICC) of NAB1 (red), FLAG (green), and DAPI (blue) in WT U2OS cells. NAB2 was cytoplasmic and nuclear while FLAG staining was absent.

Article Snippet: Antibody , STAT6 , Santa Cruz Biotechnology , Cat#sc-374021 , WB, 1:1000, IF, 1:500, ChIP.

Techniques: Control, Expressing, Liquid Chromatography with Mass Spectroscopy, Generated, FLAG-tag, Western Blot, Protein-Protein interactions, Immunocytochemistry, Staining

Journal: eLife

Article Title: NAB2-STAT6 drives an EGR1-dependent neuroendocrine program in solitary fibrous tumors

doi: 10.7554/eLife.98072

Figure Lengend Snippet: ( a ) Ranking of TCGA tumors by their average single sample gene set enrichment analysis (ssGSEA) score using the SFT gene signature of upregulated genes from . (n=2429). Neuroendocrine tumors highly express the signature while leukemias downregulate the signature. ( b ) Kaplan-Meier curve showing survival analysis of tumors in the TCGA database stratified by high or low expression of SFT gene signature of upregulated genes from (n=2429). ( c ) ssGSEA of 87 mesotheliomas from TCGA using SFT gene signature of upregulated genes from (n=2429). Shows significant upregulation of the SFT gene signature in the TCGA SH A7BH sample. ( d ) NAB2-STAT6 gene fusion in (NAB2 exons 1–6, STAT6 exons 17–22) present in TCGA SH A7BH, originally diagnosed mesothelioma. Graphic generated with Arriba, a fusion detection algorithm. ( e ) InterPro domain analysis of the top 400 most upregulated genes in SFTs shows Homeobox and Cadherin as the most significantly enriched protein domains. ( f ) Screenshot displays HOXA locus which has significant RNAPII localization in SFTs indicating that the Homeobox genes are actively transcribed in SFTs. ( g ) Motif analysis of 11155 distal enhancer (1>kb from nearest TSS) RNAPII peaks using HOMER shows HOX, GRE, and PGR as the most significantly enriched TF matrices.

Article Snippet: Antibody , STAT6 , Santa Cruz Biotechnology , Cat#sc-374021 , WB, 1:1000, IF, 1:500, ChIP.

Techniques: Expressing, Generated

Journal: eLife

Article Title: NAB2-STAT6 drives an EGR1-dependent neuroendocrine program in solitary fibrous tumors

doi: 10.7554/eLife.98072

Figure Lengend Snippet: Model for NAB2-STAT6’s function; NAB2-STAT6 is directed to the nucleus by its STAT6 moiety. The fusion protein then directs co-activators NAB1, NAB2, and EGR1 to the nucleus which in turn directs NAB2-STAT6 to EGR1 target promoters and enhancers which are highly activated by the complex of co-activators recruited. This figure was created using BioRender.com .

Article Snippet: Antibody , STAT6 , Santa Cruz Biotechnology , Cat#sc-374021 , WB, 1:1000, IF, 1:500, ChIP.

Techniques:

Journal: Nature Communications

Article Title: c-Myc inactivation of p53 through the pan-cancer lncRNA MILIP drives cancer pathogenesis

doi: 10.1038/s41467-020-18735-8

Figure Lengend Snippet: a c-Myc bound to MILIP promoter in A549 and MCF-7 cells. An E-box motif, not associated with MYC target genes on Chr22, was used as a negative control. Data shown represent three independent experiments. ChIP, chromatin immunoprecipitation. b c-Myc silencing downregulated MILIP expression in A549 and MCF-7 cells. c-Myc responsive lncRNA OVAAL was used as a positive control. Data are mean ± s.d.; n = 3 independent experiments, one-way ANOVA followed by Tukey’s multiple comparisons test. c c-Myc silencing reduced the activity of reporters with intact c-Myc binding region (BR) of MILIP promoter but not that with the c-Myc-BR deleted (ΔBR) in A549 and MCF-7 cells. Data are mean ± s.d.; n = 3 independent experiments, two-tailed Student’s t test. d Representative microscopic photographs of in situ hybridization (ISH) analysis of MILIP expression in formalin-fixed paraffin-embedded (FFPE) lung adenocarcinoma (LUAD; n = 88 biologically independent samples) compared with paired adjacent normal tissues. Scale bar, 5 µm. e Quantitation of MILIP expression as detected in ( d ) in FFPE LUAD in comparison with paired adjacent normal tissues. RS, reactive score. Two-tailed Student’s t test. f Ingenuity Pathway Analysis (IPA) of RNA-seq data showing that p53 signaling was the most enriched pathway in A549 cells transfected with a MILIP siRNA (si-MILIP.2) relative to those introduced with the control siRNA. Orange bars represent pathways that were activated, and blue bars, inactivated. DEGs differentially expressed genes. g c-Myc knockdown upregulated p53 expression, which was diminished by MILIP overexpression in A549 and MCF-7 cells. Data are representatives or mean ± s.d.; n = 3 independent experiments, One-way ANOVA followed by Tukey’s multiple comparisons test. h c-Myc overexpression downregulated p53 expression, which was abolished by knockdown of MILIP in A549 and MCF-7 cells. Data shown represent three independent experiments. DOX, doxycycline, 200 ng/mL. Source data of Fig. 1a–c, e–h are provided as a Source Data file.

Article Snippet: P53 protein concentrations were measured using PathScan Total p53 Sandwich ELISA Kit (Cell Signaling Technology, Cat#7370C), and Recombinant Human TP53 protein (Creative Biomart, Cat#TP53-15H) was used as a standard.

Techniques: Negative Control, Chromatin Immunoprecipitation, Expressing, Positive Control, Activity Assay, Binding Assay, Two Tailed Test, In Situ Hybridization, Formalin-fixed Paraffin-Embedded, Quantitation Assay, Comparison, RNA Sequencing, Transfection, Control, Knockdown, Over Expression

Journal: Nature Communications

Article Title: c-Myc inactivation of p53 through the pan-cancer lncRNA MILIP drives cancer pathogenesis

doi: 10.1038/s41467-020-18735-8

Figure Lengend Snippet: a Induced knockdown of MILIP by DOX reduced cancer cell anchorage-independent growth. Scale bar, 50 µm. Data are representatives or mean ± s.d.; n = 3 independent experiments, two-tailed Student’s t test. DOX: 200 ng/mL. b Induced knockdown of MILIP by DOX retarded A549 xenograft growth, which was reversed by DOX withdrawal in nu/nu mice. Data are mean ± s.d.; n = 6 mice per group, one-way ANOVA followed by Tukey’s multiple comparison test. DOX: 2 mg/mL supplemented with 10 mg/mL sucrose in drinking water. c MILIP silencing reduced A549 cell clonogenicity, which was attenuated by co-silencing of p53 siRNA1. Scale bar, 1 cm. Data are representatives or mean ± s.d.; n = 3 independent experiments, one-way ANOVA followed by Tukey’s multiple comparison test. d Kaplan–Meier analysis of the probability of overall survival (OS) of lung adenocarcinoma (LUAD; n = 477) and breast invasive carcinoma (BRCA; n = 1197) derived from the TCGA using the median/quartile of MILIP levels as the cut-off. e MILIP expression was increased in colon adenomas ( n = 15) compared with normal colon epithelia ( n = 16). Data are mean ± s.d.; two-tailed Student’s t test. f MILIP silencing decelerated anchorage-independent growth of HME-1 human mammary epithelial cells caused by c-Myc overexpression along with knockdown of p14 ARF . Scale bar, 50 µm. Data are representatives or mean ± s.d.; n = 2 independent experiments. g MILIP silencing decelerated anchorage-independent growth of MCF10A human mammary epithelial cells that did not express p14 ARF caused by c-Myc overexpression. Scale bar, 50 µm. Data are representatives or mean ± s.d.; n = 2 independent experiments. h MILIP overexpression caused anchorage-independent growth of MCF10A cells. Scale bar, 50 µm. Data are representatives or mean ± s.d.; n = 2 independent experiments. Source data of Fig. are provided as a Source Data file.

Article Snippet: P53 protein concentrations were measured using PathScan Total p53 Sandwich ELISA Kit (Cell Signaling Technology, Cat#7370C), and Recombinant Human TP53 protein (Creative Biomart, Cat#TP53-15H) was used as a standard.

Techniques: Knockdown, Two Tailed Test, Comparison, Derivative Assay, Expressing, Over Expression

Journal: Nature Communications

Article Title: c-Myc inactivation of p53 through the pan-cancer lncRNA MILIP drives cancer pathogenesis

doi: 10.1038/s41467-020-18735-8

Figure Lengend Snippet: a Induced knockdown of MILIP prolonged the half-life time of p53 protein in cycloheximide (CHX)-chase assays. Data are representatives or mean ± s.d.; n = 3 independent experiments, two-tailed Student’s t test. CHX: 40 µg/mL, DOX: 200 ng/mL. b MILIP silencing reduced p53 polyubiquitination. Data shown represent three independent experiments. c Induced knockdown of MILIP did not reduce the binding between p53 and MDM2. Data shown represent three independent experiments. d MILIP was coprecipitated with p53 using RNA immunoprecipitation (RIP) assays. Data shown represent three independent experiments. e p53 was co-pulled down with MILIP by antisense probes against MILIP using RNA pulldown assays. Data shown represent three independent experiments. f In vitro transcribed MILIP was coprecipitated with purified Flag-tagged p53 in a cell-free system using RNA immunoprecipitation (RIP) assays. Data shown represent three independent experiments. g Overexpression of MILIP but not a MILIP mutant with the −966/−1199 segment deleted (MILIP Δ−966/−1199) downregulated both endogenous (left) and overexpressed exogenous (right) p53 protein levels. Data are mean ± s.d.; n = 3 independent experiments, one-way ANOVA followed by Tukey’s multiple comparisons test. Source data of Fig. are provided as a Source Data file.

Article Snippet: P53 protein concentrations were measured using PathScan Total p53 Sandwich ELISA Kit (Cell Signaling Technology, Cat#7370C), and Recombinant Human TP53 protein (Creative Biomart, Cat#TP53-15H) was used as a standard.

Techniques: Knockdown, Two Tailed Test, Binding Assay, RNA Immunoprecipitation, In Vitro, Purification, Over Expression, Mutagenesis

Journal: Nature Communications

Article Title: c-Myc inactivation of p53 through the pan-cancer lncRNA MILIP drives cancer pathogenesis

doi: 10.1038/s41467-020-18735-8

Figure Lengend Snippet: a Knockdown of MILIP increased modification of p53 by SUMO-2/3, which was abolished by co-knockdown of TRIML2. Data shown represent three independent experiments. MG132: 10 µM. b TRIML2 overexpression increased p53 SUMOylation, which was diminished by co-overexpression of MILIP. Data shown represent three independent experiments. MG132: 10 µM. c Knockdown of MILIP upregulated p53, which was diminished by co-knockdown of TRIML2. Data shown represent three independent experiments. d Knockdown of MILIP-reduced p53 polyubiquitination, which was reversed by TRIML2 co-knockdown. Data shown represent three independent experiments. MG132: 10 µM. e MILIP overexpression increased p53 polyubiquitination, which was abolished by overexpression of TRIML2. Data shown represent three independent experiments. MG132: 10 µM. f Overexpression of TRIML2 upregulated p53. Data shown represent three independent experiments. g Knockdown of MILIP increased the amount of TRIML2 bound to p53. Data shown represent three independent experiments. IP immunoprecipitation. The proteasome inhibitor MG132 was used at 10 µM. h Knockdown of TRIML2 increased the amount of MILIP bound to p53. Data are representatives or mean ± s.d.; n = 3 independent experiments, two-tailed Student’s t test. Source data of Fig. are provided as a Source Data file.

Article Snippet: P53 protein concentrations were measured using PathScan Total p53 Sandwich ELISA Kit (Cell Signaling Technology, Cat#7370C), and Recombinant Human TP53 protein (Creative Biomart, Cat#TP53-15H) was used as a standard.

Techniques: Knockdown, Modification, Over Expression, Immunoprecipitation, Two Tailed Test

Journal: Nature Communications

Article Title: c-Myc inactivation of p53 through the pan-cancer lncRNA MILIP drives cancer pathogenesis

doi: 10.1038/s41467-020-18735-8

Figure Lengend Snippet: a , d Knockdown of c-Myc increased modification of p53 by SUMO-2/3, which was reversed by overexpression of MILIP. Data shown represent three independent experiments. MG132: 10 µM. b , e Knockdown of c-Myc decreased p53 polyubiquitination, which was reversed by overexpression of MILIP. Data shown represent three independent experiments. MG132: 10 µM. c , f Knockdown of c-Myc prolonged the half-life time of p53 protein, which was reversed by overexpression of MILIP in CHX-chase assays. Data are representatives or mean ± s.d.; n = 3 independent experiments, one-way ANOVA followed by Tukey’s multiple comparisons test. CHX: 40 µg/mL. Source data of Fig. are provided as a Source Data file.

Article Snippet: P53 protein concentrations were measured using PathScan Total p53 Sandwich ELISA Kit (Cell Signaling Technology, Cat#7370C), and Recombinant Human TP53 protein (Creative Biomart, Cat#TP53-15H) was used as a standard.

Techniques: Knockdown, Modification, Over Expression

Journal: Advanced Science

Article Title: LncRNA LIMp27 Regulates the DNA Damage Response through p27 in p53‐Defective Cancer Cells

doi: 10.1002/advs.202204599

Figure Lengend Snippet: Identification of LIMp27 as an E2F1‐responsive lncRNA that selectively supports the viability of p53‐defective cancer cells. a) LIMp27 is upregulated in COAD compared with corresponding normal tissues as revealed by analysis of the lncRNA expression data in the TCGA dataset. Data are mean ± s.d.; two‐tailed Student's t ‐test. N: normal tissues; T: tumor tissues. b) Kaplan–Meier analysis of the probability of overall survival of COAD patients ( n = 371) derived from the TCGA using the median of LIMp27 levels as the cut‐off. c) Representative microphotographs and quantitation of in situ hybridization (ISH) analysis of LIMp27 expression in formalin‐fixed paraffin‐embedded (FFPE) COAD tissues ( n = 77 biologically independent samples) compared with corresponding paired adjacent normal tissues. Scale bar, 20 µm. RS: reactive score. Two‐tailed Student's t ‐test. d) qPCR analysis showing that LIMp27 was generally more abundant in colon cancer cell lines than in the normal colon epithelial cell line FHC. Data are mean ± s.d.; n = 3 independent experiments, two‐tailed Student's t ‐test. e) Absolute quantitation of LIMp27 in HT‐29 and HCT116 COAD cell lines and the normal colon epithelial cell line FHC using qPCR. Data are mean ± s.d.; n = 3 independent experiments, two‐tailed Student's t ‐test. f) Comparison of LIMp27 expression between normal colon mucosa, colon adenoma, and colon cancer tissues derived from R2 public dataset. Data are mean ± s.d.; one‐way ANOVA followed by Tukey's multiple comparisons test. g) Chromatin immunoprecipitation (ChIP) analysis of the association between endogenous E2F1 and the E2F1‐binding motifs at the promoter of LIMp27 in HT‐29 and WiDr cell lines. Data are representatives of three independent experiments. h) SiRNA knockdown of E2F1 reduced the transcriptional activity of a LIMp27 promoter reporter construct (pGL3‐LIMp27 promoter). Data are mean ± s.d.; n = 3 independent experiments, two‐tailed Student's t ‐test. i) E2F1 silencing downregulated LIMp27 expression in HT‐29 and WiDr cell lines. Data are representatives or mean ± s.d.; n = 3 independent experiments, one‐way ANOVA followed by Tukey's multiple comparisons test. j–l) SiRNA knockdown of LIMp27 (j) inhibited cell proliferation (k) and clonogenicity (l) in HT‐29 and WiDr (p53 mutant) but not in HCT116 and RKO (p53 WT) cell lines. Data are mean ± s.d. or representatives; n = 3 independent experiments, one‐way ANOVA followed by Tukey's multiple comparison test. Scale bar, 1 cm. m,n) Kaplan–Meier analysis of the probability of overall survival of COAD patients with tumors carrying mutant p53 (m) and wild‐type p53 tumors (n) derived from the TCGA using the median of LIMp27 levels as the cut‐off.

Article Snippet: The human normal colon epithelial cell line FHC (ATCC CRL‐1831), the human COAD cell lines HT‐29 (ATCC HTB‐38), WiDr (ATCC CCL‐218), Caco‐2 (ATCC HTB‐37), HCT116 (ATCC CCL‐247), RKO (ATCC CRL‐2577), SW480 (ATCC CCL‐228), human breast cancer cell lines MCF‐7 (ATCC HTB‐22), MDA‐MB‐231 (ATCC HTB‐26), human non‐small cell lung cancer cell lines A549 (ATCC CCL‐185), H226 (ATCC CRL‐5826), and human embryonic kidney cell line HEK293T were from American Type Culture Collection (ATCC).

Techniques: Expressing, Two Tailed Test, Derivative Assay, Quantitation Assay, In Situ Hybridization, Formalin-fixed Paraffin-Embedded, Comparison, Chromatin Immunoprecipitation, Binding Assay, Knockdown, Activity Assay, Construct, Mutagenesis