Journal: Nature Communications

Article Title: CEACAM1 as a mediator of B-cell receptor signaling in mantle cell lymphoma

doi: 10.1038/s41467-025-60208-3

Figure Lengend Snippet: a Left panel. Log 2 fold-change of sgRNA counts between days 14 and 0 ( P < 0.01; two-sided permutation test) are plotted against the Z ratio of mRNA expression between MCL and naïve-B cells ( P < 0.05; two-sided t -test). Right panel. Overlap of selected genes depleted at least twofold and overexpressed in MCL with pan-dependent genes and genes associated with poor prognosis . Numbers in green boxes indicate filtering strategy (see text). b Shown are mean values of selected normalized sgRNA counts on days 0 and 14 from two biological replicates. c Immunoblots show CEACAM1 knockout in JEKO-1 cells transduced with control (gNTC) or CEACAM1 gRNAs (gCC1) followed by anti-IgM antibody stimulation (2 μg/ml, 5 min). d JEKO-1 cells were transduced with indicated sgRNAs, and viable, GFP + -transduced cells were monitored over time by FACS. Shown are the means of GFP+ fractions compared to day-2 samples from three biological replicates. Error bars, SD. * P < 0.05 by a two-sided, paired t -test. e Immunoblots show CEACAM1 knockdown by shRNA in JEKO-1 cells. f Indicated cells were transduced with CEACAM1 shRNA and viable, propidium iodide (PI)-negative cells were assessed by FACS over time. Shown are the means of PI-negative fractions compared to day-2 samples from three biological replicates. Error bars, SD. * P < 0.05, ** P < 0.01 by a two-sided, paired t -test. g CEACAM1 is required for MCL survival in vivo. JEKO-1 cells were transduced with control or CEACAM1 shRNA and intravenously transplanted into NSG mice. Shown are weekly bioluminescence images of six mice/group. h Line graphs show the means of bioluminescence signals measuring tumor growth in mice described in ( g ). Error bars, SEM. * P < 0.05 by a one-sided t -test. i Kaplan–Meier survival analysis of mice shown in ( g ). P value, log - rank test. j Top panel, Generation of double SOX11/CCND1 transgenic (DT) and CEACAM1-deficient mice. Middle panel, Representative FACS plots show MCL-like population (CD19 + CD5 + CD23-). Bottom panel, bar graphs show means of %MCL-like cells from the peripheral blood of indicated mice and sample sizes. Error bars, SD. P values, two-sided unpaired t -test. Source data are provided as a Source Data file.

Article Snippet: To validate the screen results, top- and bottom-strand oligos of control (gNTC) or the top three CEACAM1 gRNAs were synthesized and cloned into the library backbone plasmid lentiGuide-Puro (Addgene #52963) using a Golden Gate assembly as described .

Techniques: Expressing, Western Blot, Knock-Out, Transduction, Control, Knockdown, shRNA, In Vivo, Transgenic Assay

Journal: Nature Communications

Article Title: CEACAM1 as a mediator of B-cell receptor signaling in mantle cell lymphoma

doi: 10.1038/s41467-025-60208-3

Figure Lengend Snippet: a – c Box plots show CEACAM1 mRNA expression levels in MCL and other cell types from the datasets GSE2350 ( a , b ) and GSE132929 ( c ). The boxes extend from the 25th to 75th percentiles with the center line as median and whiskers drawn from 10th to 90th percentiles. **** P < 0.0001 by a two-sided Mann–Whitney U - test. GC germinal center, Mem memory, Fo follicular, BL Burkitt lymphoma, DLBCL diffuse large B-cell lymphoma, FL follicular lymphoma, PEL primary effusion lymphoma. d Representative immunoblot analysis of CEACAM1 expression in indicated cells from at least three independent experiments. Numbers below bands represent densitometric values of CEACAM1 signals normalized over GAPDH loading controls. e Flow cytom e try analysis of surface CEACAM1 expression in MCL PDXs compared to CEACAM1-negative RAMOS cells from at least two independent experiments. Live cell gating in this experiment and throughout the study is described in Supplementary Fig. . f Representative immunohistochemistry images showing varying CEACAM1 staining levels for indicated formalin-fixed paraffin-embedded tissue microarrays from three independent experiments. In cHL tissue, background plasma cells are also stained positive. cHL classic Hodgkin lymphoma, CLL chronic lymphocytic leukemia. See Table for a summary of CEACAM1 positivity in specific diseases. Source data are provided as a Source Data file.

Article Snippet: To validate the screen results, top- and bottom-strand oligos of control (gNTC) or the top three CEACAM1 gRNAs were synthesized and cloned into the library backbone plasmid lentiGuide-Puro (Addgene #52963) using a Golden Gate assembly as described .

Techniques: Expressing, MANN-WHITNEY, Western Blot, Immunohistochemistry, Staining, Formalin-fixed Paraffin-Embedded, Clinical Proteomics

Journal: Nature Communications

Article Title: CEACAM1 as a mediator of B-cell receptor signaling in mantle cell lymphoma

doi: 10.1038/s41467-025-60208-3

Figure Lengend Snippet: a – e Indicated cell lines were transduced with control or CEACAM1 shRNA, followed by stimulation with anti-IgM (1 μg/mL). a Shown are normalized Ca 2+ signals. **** P < 0.0001 by two-way ANOVA. Data were representative of at least three independent experiments. b , c Immunoblots show effects of CEACAM1 knockdown on BCR signaling components. Data were representative of three independent experiments. d , e Immunoblots show CEACAM1 knockdown (top panels) and its effect on Ca 2+ signals (bottom panels). f Top panel, immunoblots of splenocytes from wild type (+/+) or Ceacam1-deficient (−/−) mouse with indicated antibodies. Bottom panel, Ca 2+ signals of indicated splenic B220 + B cells. Data from ( d – f ) are representative of at least two independent experiments. g CEACAM1 mRNA expression levels in CD19+ sorted MCL cells from peripheral blood (PB) or lymph nodes (LN) analyzed from the GSE70910 dataset . Closed circles or squares represent individual patient samples. Horizontal bars from each group indicate mean mRNA expression. P value is from a two-sided unpaired t -test with Welch’s correction. h CEACAM1 expression is correlated with ibrutinib response. Left panels, FACS plots showing surface CEACAM1 expression on indicated MCL and MZL cell lines. Isotype, negative isotype antibody on Z-138 cells. Right panels, Indicated cell lines were treated with indicated doses of ibrutinib for 4 days, and viable propidium iodide (PI)-negative cells were assessed by flow cytometry. Line graphs showing means of normalized PI-negative fractions from three independent experiments. Error bars, SD. Fifty-percent inhibition concentration (IC 50 ) values were calculated by GraphPad Prism v8. i , j ITIM tyrosine residues are required for BCR signaling. JEKO-1 cells transduced with control (gNTC) or CEACAM1 gRNA (gCC1), followed by reintroducing WT CEACAM1 (4 L), CC1-4L-Y493F/Y520F mutant (YY/FF), or short cytoplasmic tail (4S). Controls or reconstituted cell lines were verified for CEACAM1 expression by FACS using B1.1 antibody ( i ) and stimulated with 1 μg/mL of anti-IgM for 5 min, followed by immunoblotting with indicated antibodies, including the CEACAM1 E1 antibody ( j ). Data from ( i , j ) are representative of at least two independent experiments. Source data are provided as a Source Data file.

Article Snippet: To validate the screen results, top- and bottom-strand oligos of control (gNTC) or the top three CEACAM1 gRNAs were synthesized and cloned into the library backbone plasmid lentiGuide-Puro (Addgene #52963) using a Golden Gate assembly as described .

Techniques: Transduction, Control, shRNA, Western Blot, Knockdown, Expressing, Flow Cytometry, Inhibition, Concentration Assay, Mutagenesis

Journal: Nature Communications

Article Title: CEACAM1 as a mediator of B-cell receptor signaling in mantle cell lymphoma

doi: 10.1038/s41467-025-60208-3

Figure Lengend Snippet: a Diagram of sucrose-density gradient fractionation of lipid rafts. b Left panels, Immunoblots of fractions indicated in ( a ) from control or CEACAM1-knockout JEKO-1 cells stimulated with control or anti-IgM antibody (1 μg/ml) for 2 min. Right panels, immunoblot signal quantification of fraction I (red dashed-line box) after normalization to Flotillin-1 and unstimulated controls. Shown are the means of fold changes from three independent experiments. Error bars, SD. ** P < 0.01, * P < 0.05 by a two-sided, paired t -test. ns not significant. c Left panels, Control (gNTC) or CEACAM1 knockout (gCC1) JEKO-1 cells were stimulated with 2 μg/ml anti-IgM antibody for 2 min. Shown are representative cells from confocal immunofluorescence images in Supplementary Fig. of control and IgM-stimulated cells co-stained with anti-FLNA (green) and anti-LYN (red) antibodies, followed by nuclear staining with DAPI (blue). Scale bar, 2 μm. Right panels, quantified fluorescent signals for each cell from the samples described. Shown are the sums of intensities in signal-positive areas per cell in arbitrary units from three independent experiments. Horizontal red bars indicate the mean. Approximately 200 cells from each sample were analyzed. **** P < 0.0001 by a two-sided unpaired t -test. ns not significant. d CEACAM1 is required for lipid-raft assembly. Top panels, Control, CEACAM1 knockout (CC1 KO), or CC1 KO + CEACAM1-4L (4 L) JEKO-1 cells were stimulated with 2 μg/ml anti-IgM antibody for 5 min. Shown are representative confocal immunofluorescence images of control and IgM-stimulated cells stained with anti-CEACAM1 antibody (green), GM1 via cholera toxin B (red), p-SRC Y416 antibody (green), or F-actin via Actin-Stain 555 Phalloidin (red) followed by nuclear staining with DAPI (blue). Scale bar, 10 μm. Bottom panels, quantified fluorescent signals from the samples described in the top panels. Shown are the sums of intensities in signal-positive areas per cell in arbitrary units from three independent experiments. Horizontal red bars indicate the mean. Approximately 200 cells from each sample were analyzed. **** P < 0.0001 by a two-sided unpaired t -test. Source data are provided as a Source Data file.

Article Snippet: To validate the screen results, top- and bottom-strand oligos of control (gNTC) or the top three CEACAM1 gRNAs were synthesized and cloned into the library backbone plasmid lentiGuide-Puro (Addgene #52963) using a Golden Gate assembly as described .

Techniques: Fractionation, Western Blot, Control, Knock-Out, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: CEACAM1 as a mediator of B-cell receptor signaling in mantle cell lymphoma

doi: 10.1038/s41467-025-60208-3

Figure Lengend Snippet: a , b Top panels, Proximity ligation assay (PLA) showing interactions (visualized as red dots using Airyscan FAST 2D confocal microscope and a 40x/1.2NA water objective) between CEACAM1 (CC1) and the indicated proteins in JEKO-1 and MINO cells stimulated with 2 μg/ml of anti-IgM antibody for the indicated times. Bottom panels, Quantification of PLA signals shown in the top panels for ~200 cells on average from three independent experiments using QuPath 0.3.2 software. **** P < 0.0001, *** P < 0.001 by a two-tailed unpaired t -test. ns not significant. c , d Immunoprecipitation analysis of CEACAM1 interactions. Top panels, JEKO-1 or MINO cells were stimulated with 2 μg/ml of anti-IgM antibody for the indicated times, and CEACAM1 was immunoprecipitated with a CEACAM1-specific antibody or IgG control antibodies, followed by immunoblotting with the indicated antibodies. One percent of the total lysates was used as an input control. Bottom panels, Quantification of indicated co-IP signals shown in the top panels. Bar graphs show the means and individual densitometric values from three independent experiments for each timepoint normalized to the CEACAM1 pull-down signals. Error bars, SD. *** P < 0.001, ** P < 0.01, * P < 0.05 by a two-sided unpaired t -test. ns not significant. Source data are provided as a Source Data file.

Article Snippet: To validate the screen results, top- and bottom-strand oligos of control (gNTC) or the top three CEACAM1 gRNAs were synthesized and cloned into the library backbone plasmid lentiGuide-Puro (Addgene #52963) using a Golden Gate assembly as described .

Techniques: Proximity Ligation Assay, Microscopy, Software, Two Tailed Test, Immunoprecipitation, Control, Western Blot, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: CEACAM1 as a mediator of B-cell receptor signaling in mantle cell lymphoma

doi: 10.1038/s41467-025-60208-3

Figure Lengend Snippet: a Left panel, Immunoprecipitation analysis of CEACAM1 interactions. CEACAM1-knockout JEKO-1 cells were transduced with either full-length (4 L) or N-domain truncated (ΔN) CEACAM1 constructs, stimulated with 2 μg/ml anti-IgM antibody for the indicated times, followed by CEACAM1 immunoprecipitation and immunoblotting with the indicated antibodies. One percent of the total lysates was used as an input control. p-Y, anti-phosphotyrosine antibody clone 4G10. Right panels, Quantification of indicated co-IP signals shown in the left panels. Bar graphs show the means and individual densitometric values from four independent experiments for each timepoint normalized to the CEACAM1 pull-down signals. Error bars, SD. ** P < 0.01, * P < 0.05 by two-way ANOVA. b Proximity ligation assay (PLA) showing interactions (visualized as red dots using Airyscan FAST 2D confocal microscope and a 40x/1.2NA water objective) between 4 L or ΔN and the indicated proteins. c Quantitation of PLA signals shown in ( b ) for 100–300 cells on average from three independent experiments using QuPath 0.3.2 software. Error bars indicate means with S.D. **** P < 0.0001 by two-sided Mann–Whitney U -test. ns not significant. Source data are provided as a Source Data file.

Article Snippet: To validate the screen results, top- and bottom-strand oligos of control (gNTC) or the top three CEACAM1 gRNAs were synthesized and cloned into the library backbone plasmid lentiGuide-Puro (Addgene #52963) using a Golden Gate assembly as described .

Techniques: Immunoprecipitation, Knock-Out, Transduction, Construct, Western Blot, Control, Co-Immunoprecipitation Assay, Proximity Ligation Assay, Microscopy, Quantitation Assay, Software, MANN-WHITNEY

Journal: Nature Communications

Article Title: CEACAM1 as a mediator of B-cell receptor signaling in mantle cell lymphoma

doi: 10.1038/s41467-025-60208-3

Figure Lengend Snippet: a Left panel, CEACAM1 suppresses BCR signaling in Z-138 cells. Z-138 cells were transduced with control empty vector (EV), WT CEACAM1 (CC1-4L), or short cytoplasmic tail CEACAM1 (CC1-4S). Transduced cells were stimulated with 2 μg/mL of anti-IgM F(ab’)2 fragments for the indicated times, followed by immunoblot analysis probed with the indicated antibodies. HA, hemagglutinin, a protein tag in-framed with CEACAM1 to detect the 4S isoform. Right panel, Quantification of the immunoblot signals shown in the left panel. Bar graphs show the means of densitometric values from the indicated time points normalized to GAPDH loading controls from two independent experiments. P values, one-sided permutation test. b , c CEACAM1-knockout JEKO-1 cells (gCC1) or Z-138 cells were transduced with either empty vector control or full-length (4 L) CEACAM1 construct, stimulated with 2 μg/ml anti-IgM antibody for the indicated times, followed by CEACAM1 immunoprecipitation and immunoblotting with indicated antibodies. One percent of the total lysates was used as an input control ( b ). The samples shown in ( b ) derive from the same experiment, but different gels for CEACAM1, p-SYK Y352 , SYK, SHP-1, GAPDH, and another for SHP-2 were processed in parallel. Bar graphs show the quantification of the co-IP signals shown in ( c ). Shown are the means of densitometric values from two independent experiments for each timepoint normalized to the CEACAM1 pull-down signals. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05 by two-way ANOVA. d Left panels, Proximity ligation assay (PLA) showing interactions (visualized as red dots using Airyscan FAST 2D confocal microscope and a 40x/1.2NA water objective) between CEACAM1 (CC1) and the indicated proteins in Z-138 cells or Z-138 cells transduced with CEACAM1-4L followed by stimulation with 2 μg/ml of anti-IgM antibody for the indicated times. Right panels, Quantification of PLA signals shown in the right panels for ~200 cells on average from three independent experiments using QuPath 0.3.2 software. **** P < 0.0001 by a two-sided unpaired t -test with Welch’s correction. Source data are provided as a Source Data file.

Article Snippet: To validate the screen results, top- and bottom-strand oligos of control (gNTC) or the top three CEACAM1 gRNAs were synthesized and cloned into the library backbone plasmid lentiGuide-Puro (Addgene #52963) using a Golden Gate assembly as described .

Techniques: Transduction, Control, Plasmid Preparation, Western Blot, Knock-Out, Construct, Immunoprecipitation, Co-Immunoprecipitation Assay, Proximity Ligation Assay, Microscopy, Software

Journal: Nature Communications

Article Title: CEACAM1 as a mediator of B-cell receptor signaling in mantle cell lymphoma

doi: 10.1038/s41467-025-60208-3

Figure Lengend Snippet: Following antigen stimulation in B-cell lymphomas with abundant CEACAM1 expression, CEACAM1 binds to FLNA, which anchors to the actin cytoskeleton and lipid rafts, and recruits SYK to the proximity of CD79A/B to enhance BCR activity. In cells with low or no CEACAM1 expression (and potentially low SYK expression), SHP-1 and SHP-2 outcompete for CEACAM1 binding, leading to signal attenuation. Created in BioRender. Ngo, V. (2025) https://BioRender.com/dl16wxh .

Article Snippet: To validate the screen results, top- and bottom-strand oligos of control (gNTC) or the top three CEACAM1 gRNAs were synthesized and cloned into the library backbone plasmid lentiGuide-Puro (Addgene #52963) using a Golden Gate assembly as described .

Techniques: Expressing, Activity Assay, Binding Assay

Journal: Cancer Immunology Research

Article Title: Tumor Cell–Intrinsic Decr2 Regulates Ferroptosis and Immunotherapy Efficacy

doi: 10.1158/2326-6066.CIR-24-0519

Figure Lengend Snippet: Decr2 expression correlates with patient outcomes, and Slc7a11 is a candidate therapeutic target. A, Fold changes of Decr2 in matched pretherapy and on-therapy samples from patients with melanoma. B, Relationship between overall survival and mRNA expression level of Decr2 in human patients treated with ipilimumab. C, Decr2 deletion (deep and shallow) and Decr2 copy-number gain association with overall survival in response to ipilimumab. D, IF staining of human melanoma tissue sections. DECR2 (green), SOX10 (red), and DAPI (blue). Scale bar, 50 μm. E, Percentage of Decr2 + Sox10 + cells and relative intensity of DECR2 expression in tissues of patients with melanoma: pretreatment or on-treatment with anti–PD-1. F, KD of Slc7a11 in Decr2 -deficient tumor cells restores anti–PD-L1 efficacy. Control shRNA–infected tumors (in black), Decr2 single KD tumors (in red, labeled as Decr2 KD), and Decr2 Slc7a11 double KD (in blue, labeled as Decr2 Slc7a11 DKD). n ≥ 9 mice per group. G, The end timepoint of tumor area with or without anti–PD-L1 treatment. Control shRNA–infected tumors (in black), Decr2 single KD tumors (in red, labeled as Decr2 KD), Decr2 Slc7a11 double KD (in blue, labeled as Decr2 Slc7a11 DKD). P values are determined using the Mann–Whitney test ( A ), the two-tailed t test ( C, E, G ), and two-way ANOVA ( F ). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. CR, complete responder; PD, progressive disease; PR, partial responder; SD, stable disease.

Article Snippet: An IF protocol was developed using antibodies specific to DECR2 (Abcam, ab153849, 1/50 dilution) and SOX10 (R&D Systems, MAB2864, 1/200 dilution) on formalin-fixed, paraffin-embedded tissue sections.

Techniques: Expressing, Staining, Control, shRNA, Infection, Labeling, MANN-WHITNEY, Two Tailed Test

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: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: ID family protein expression and regulation in hypoxic pulmonary hypertension

doi: 10.1152/ajpregu.00866.2009

Figure Lengend Snippet: Localization of ID2 expression in peripheral pulmonary vessels of wild-type and Id1 null mouse lungs. A–P: immunofluorescence staining for ID2 using rabbit monoclonal antibody clone 98–2 (red) and mouse monoclonal anti-α-SMA (green) in small (20- to 50-μm) muscularized peripheral pulmonary vessels in wild-type (A–D, I–L) and Id1 null mice (E–H, M–P) under normoxic conditions (A–H) and after 1 wk of hypoxia (I–P). Nuclear staining with DAPI is shown in blue. Q: negative staining with rabbit IgG control in wild-type normoxic lungs. R and S: quantification of cell-specific ID2 indexes in EC (R) and VSMC (S) compartments in peripheral vessels from normoxic and 1-wk hypoxic wild-type and Id1 null mouse lungs, as indicated. Arrows indicate nuclear staining in VSMCs; arrowheads, ECs. Scale bar = 100 μm. Values are means ± SE (n = 4 per group). aP < 0.05 vs. corresponding genotype normoxic control by one-way ANOVA with Bonferroni correction.

Article Snippet: Immunofluorescence was performed on formalin-fixed, paraffin-embedded lung sections with the ID1–3 antibodies outlined above and detected with a rabbit IgG-specific biotin-conjugated secondary antibody, followed by streptavidin-conjugated HRP (ABC kit, Vector Laboratories).

Techniques: Expressing, Immunofluorescence, Staining, Negative Staining