anti ezh2 antibodies  (Cell Signaling Technology Inc)

Journal: JCI Insight

Article Title: Inhibition of histone methyltransferase EZH2 for immune interception of colorectal cancer in Lynch syndrome

doi: 10.1172/jci.insight.177545

Figure Lengend Snippet: ( A ) Western blot analysis was performed in the lysates from colonic mucosal stripping of VCMsh2T Hu mice ( N = 3/group): control and GSK treatment with quantification shown in bar graph below blots. Histone H3 modification was assessed using anti-H3K27me3 ( P = 0.0003), anti-H3K9me3 ( P = 0.0091), anti-H3K36me3 ( P = 0.6534), and anti-H3K4me3 ( P = 0.0005) antibodies with histone H3 as loading control. Stemness and proliferation were measured via anti-LGR5 ( P = 0.3583), anti-EPCAM ( P = 0.0001), anti-GATA3 ( P = 0.2559), and Ki67 antibodies ( P = 0.0572). Anti-EZH2 ( P = 0.3091) was probed to assess efficacy of GSK503 activity in the colonic mucosa. The loading control for each nonhistone blot was β-actin. Quantification was performed using ImageJ, and density was normalized to control samples for each probe. ( B ) IHC staining of colonic tissue from VCMsh2T Hu mice. Images shown are a single field of view (original magnification, ×20). Scale bar: 200 μm. ( C ) A representative image of sequential immunofluorescence using Lunaphore COMET platform from VCMsh2T Hu colonic mucosa ( N = 3/group) stained with DAPI (red), E-cadherin (blue), CD8 (green), Ki67 (yellow), and CD163 (white) (original magnification, ×20). ( D ) Quantification of Comet data shown in C . ( E ) EZH2 knockdown in mouse organoids phenocopied similar results obtained with GSK503 inhibition of EZH2. Quantitative gene expression analysis results demonstrated significant changes in gene expression for Cdx 2 ( P = 0.0026), Dpp4 ( P = 0.022), Epcam ( P = 0.0003), Lgr5 ( P = 0.001), and Muc2 ( P = 0.0005). The mRNA levels of Krt20 and Vill were not significant. The graphed data are expressed as mean ± SEM. For all graphs, Student’s t test was used to determine significance. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Slides were then incubated in secondary antibody (ImmPRESS secondary antibody [HRP polymer] was used depending on the primary source; goat anti-rabbit IgG was used as EZH2 primary antibody [Vector Laboratories, MP-7451]; goat anti-mouse IgG was used as Ki-67 secondary antibody [Vector Laboratories, MP-7452]) for 1 hour at room temperature and then exposed to DAB (Vector Laboratories) for 1 minute followed by Mayer’s hematoxylin counterstain for 1 minute.

Techniques: Western Blot, Stripping Membranes, Control, Modification, Activity Assay, Immunohistochemistry, Immunofluorescence, Staining, Knockdown, Inhibition, Gene Expression

Journal: Cancer Biology & Therapy

Article Title: USP44 promotes chemotherapeutic drug resistance of triple negative breast cancer through EZH2 protein stability

doi: 10.1080/15384047.2025.2529652

Figure Lengend Snippet: USP44 produces chemotherapy resistance to TNBC via EZH2. (a) BT-549 cells with or without OE-USP44 were lysed using RIPA and the lysates were pretreated with protein A/G beads at 4 C for one hour. The cell lysates were then treated with protein A/G beads containing anti-USP44 (5 µg) antibodies at 4 C overnight. Then, the supernatant was discarded and loading buffer was added to crack the magnetic beads. 10% SDS-PAGE gel was used to load 100 μg of protein and stained with Coomassie brilliant blue. Mass spectrometry showed the spectrogram of EZH2 in BT-549 cells pulled down by the USP44 antibody. (b) Expression of EZH2 in TNBC cells with or without OE-USP44/shUSP44 was detected by western blot. C) TNBC cells with OE-USP44 were treated with different concentration of DOX with or without GSK126 (2 μM) for 48 hours. Then, cell viability was measured with the CCK-8 kit ( n = 3). (d) Under the same conditions as (c), Cleaved PARP and H3K27ME3 protein was detected by western blot in two TNBC cell lines. β-actin was used as the loading control for western blot. Boxplots are shown as mean ± SD. * p < .05; ** p < .01; *** p < .001.

Article Snippet: The antibodies used in this study include mouse anti-human USP44 (Santa cruz, sc -377,203), rabbit anti-human EZH2 (Proteintech 21,800–1-AP) for co-immunoprecipitation and mouse anti-human EZH2 (Abcam, ab283270), rabbit anti-human PARP1(Proteintech 13,371–1-AP), mouse anti human UB (CST, 3936), anti-FLAG tag (sigma, F1084) and rabbit anti-human β- Actin (Abclonal, AC026).

Techniques: Magnetic Beads, SDS Page, Staining, Mass Spectrometry, Expressing, Western Blot, Concentration Assay, CCK-8 Assay, Control

Journal: Cancer Biology & Therapy

Article Title: USP44 promotes chemotherapeutic drug resistance of triple negative breast cancer through EZH2 protein stability

doi: 10.1080/15384047.2025.2529652

Figure Lengend Snippet: USP44 stabilizes EZH2 through deubiquitinase activity. (a) Extracts from MDA-MB-231 cells were isolated for co-immunoprecipitation using an anti-USP44 antibody or anti-EZH2 antibody. Specifically, MDA-MB-231 cells were lysed using RIPA and the lysates were pretreated with protein A/G beads at 4 C for one hour. The cell lysates were then treated with protein A/G beads containing anti-USP44 (5 µg) or anti-EZH2 (5 µg) antibodies at 4°C overnight. Then, the supernatant was discarded and loading buffer was added to crack the magnetic beads. The interaction of endogenous USP44 and EZH2 was tested. Normal mouse IgG was used as a control. (b) BT-549 cells with or without OE-USP44 were treated with CHX (25 µg/mL) and harvested at the indicated times (0,2,4,8 hours), then protein levels of USP44 amd EZH2 were analyzed by western blot. (c) Similarly, the same BT-549 cells with or without shUSP44 were treated as above, then protein levels of USP44 amd EZH2 were analyzed by western blot. (d) BT-549 cells with or without shUSP44 were treated with or without MG132 (1 μM) for 24 hours. The protein expression levels of USP44 and EZH2 were confirmed followed by western blot. β-actin was used as the loading control. (e) pLent-puro-ubiquitin plasmids was transfected into BT-549 cells with or without OE-USP44. After continuing to incubate for 48 h, the cells were lyzed with RIPA lysate and MG132 (10 μM) was added 2 hours before this step. Then the lysates were pretreated with protein A/G beads at 4 C for one hour. The cell lysates were then treated with protein A/G beads containing anti-EZH2 (5 µg) antibody at 4°C overnight. Then, the supernatant was discarded and loading buffer was added to crack the magnetic beads. USP44 ubiquitination was detected by western blot with anti-UB antibody. The protein expression levels of USP44 and EZH2 in BT-549 cells were confirmed. Boxplots are shown as mean ± SD. * p < .05; ** p < .01; *** p < .001.

Article Snippet: The antibodies used in this study include mouse anti-human USP44 (Santa cruz, sc -377,203), rabbit anti-human EZH2 (Proteintech 21,800–1-AP) for co-immunoprecipitation and mouse anti-human EZH2 (Abcam, ab283270), rabbit anti-human PARP1(Proteintech 13,371–1-AP), mouse anti human UB (CST, 3936), anti-FLAG tag (sigma, F1084) and rabbit anti-human β- Actin (Abclonal, AC026).

Techniques: Activity Assay, Isolation, Immunoprecipitation, Magnetic Beads, Control, Western Blot, Expressing, Ubiquitin Proteomics, Transfection

Journal: Cancer Biology & Therapy

Article Title: USP44 promotes chemotherapeutic drug resistance of triple negative breast cancer through EZH2 protein stability

doi: 10.1080/15384047.2025.2529652

Figure Lengend Snippet: In vivo validity of targeting EZH2 to sensitize TNBC cells to DOX. (a) Nude BALB/C mice were subcutaneously xenografted with 4T1cells (1×10 cells) into the flanks and injected intraperitoneally with DOX (3 mg/kg) and GSK126 (100 mg/kg) alone or in combination every two days for consecutive 14 days. (b) After 14 days, the mice were executed and the xenograft tumors were isolated. (c) Tumor weighing analysis. (d) Tumor growth curves for each group. (e) Representative IHC images showing H3K27me3 and Ki-67 expression in tumors from each group of mice. Boxplots are shown as mean ± SD. * p < .05; ** p < .01; *** p < .001.

Article Snippet: The antibodies used in this study include mouse anti-human USP44 (Santa cruz, sc -377,203), rabbit anti-human EZH2 (Proteintech 21,800–1-AP) for co-immunoprecipitation and mouse anti-human EZH2 (Abcam, ab283270), rabbit anti-human PARP1(Proteintech 13,371–1-AP), mouse anti human UB (CST, 3936), anti-FLAG tag (sigma, F1084) and rabbit anti-human β- Actin (Abclonal, AC026).

Techniques: In Vivo, Injection, Isolation, Expressing

Journal: Cancer Biology & Therapy

Article Title: USP44 promotes chemotherapeutic drug resistance of triple negative breast cancer through EZH2 protein stability

doi: 10.1080/15384047.2025.2529652

Figure Lengend Snippet: A graphic abstract of USP44-EZH2 axis regulating DOX resistance in TNBC.

Article Snippet: The antibodies used in this study include mouse anti-human USP44 (Santa cruz, sc -377,203), rabbit anti-human EZH2 (Proteintech 21,800–1-AP) for co-immunoprecipitation and mouse anti-human EZH2 (Abcam, ab283270), rabbit anti-human PARP1(Proteintech 13,371–1-AP), mouse anti human UB (CST, 3936), anti-FLAG tag (sigma, F1084) and rabbit anti-human β- Actin (Abclonal, AC026).

Techniques:

Journal: Nucleic Acids Research

Article Title: Nuclear actin-dependent Meg3 expression suppresses metabolic genes by affecting the chromatin architecture at sites of elevated H3K27 acetylation levels

doi: 10.1093/nar/gkaf280

Figure Lengend Snippet: Integration of ChIP-seq, ChIRP-seq, and ATAC-seq data sets shows increase of both active and repressive histone marks in KO-specific Meg3 ChIRP-seq peaks. Density plots and heatmaps displaying scaled-read densities for ATAC-Seq, Brg1 , Ezh2 , Suz12 , H3K9me3, H3K27me3, and H3K27ac in 3 kb regions surrounding WT-specific (top) and KO-specific (bottom) Meg3 Peaks. Scale bar shows normalized RPKM.

Article Snippet: Hundred micrograms of fragmented chromatin was mixed with 5 g of Ezh2 (D2C9) XP Rabbit mAb antibody, Cell signaling.

Techniques: ChIP-sequencing

Journal: Frontiers in Immunology

Article Title: Dual targeting of EZH2 and PD-L1 in Burkitt’s lymphoma enhances immune activation and induces apoptotic pathway

doi: 10.3389/fimmu.2025.1578665

Figure Lengend Snippet: Dose-dependent effects of iEZH2 and dEZH2 in human lymphoma cell lines. (A) Human lymphoma cell lines were treated with 1.25 μM, 2.5 μM, 5 μM and 10 μM of iEZH2 or dEZH2 for 72 hours. Cell viability was compared with control group which was treated DMSO. (B, C) The protein expression level of EZH2 and c-MYC relative to GAPDH was measured by western blot analysis. Burkitt’s lymphoma cell lines, (B) Ramos and (C) Daudi, were exposed to 10 μM of iEHZ2 or dEZH2 for 24 hours. Error bars are shown as mean ± SD. Data analyzed using two-tailed unpaired student’s t-tests. *, P ≤ 0.05; ***, P ≤ 0.001.

Article Snippet: Nonspecific background was blocked with 10% bovine serum albumin (#BSAS0.1, Bovogen, VIC, AU) in PBS for 60 min. For removal lipofuscin autofluorescence in tissues, TrueBlack (#23007, Biotium, CA, USA) was treated for 30 sec. Each tissue was stained with primary antibodies against EZH2 (#5246, Cell Signaling Technology), c-MYC (#18583, Cell Signaling Technology) CD8 (#MA1-81692, Invitrogen), granzyme B (#17215, Cell Signaling Technology), IFN-γ (#AF-285-NA, Novus Biologicals, CO, USA), and cleaved caspase-3 (#9664, Cell Signaling Technology) overnight at 4°C.

Techniques: Control, Expressing, Western Blot, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Dual targeting of EZH2 and PD-L1 in Burkitt’s lymphoma enhances immune activation and induces apoptotic pathway

doi: 10.3389/fimmu.2025.1578665

Figure Lengend Snippet: Enhanced levels of HLA class 1 and PD-L1 affected by EZH2 inhibition or IFN-γ in Burkitt’s lymphoma. (A, B) HLA class 1 expression levels were measured by flow cytometry. (A) Ramos and (B) Daudi cells were exposed to 10 μM of iEHZ2 or dEZH2, along with 10 ng/mL of human IFN-γ for 48 hours. Compared to the isotype control, HLA class 1 levels were analyzed using histogram and are shown in the graphs on the right. (C, D) Expression levels of PD-L1 were determined using flow cytometry with dot plots. PD-L1 levels were compared to the isotype control, and the analyzed data are presented in the graphs below. 10 ng/mL of human IFN-γ was treated in (C) Ramos and (D) Daudi cell lines for 48 hours, along with 10 μM of iEHZ2 or dEZH2. (E) The relative IRF-1 mRNA expression in Daudi was measured by RT-qPCR, and normalized to GAPDH. Daudi cells were exposed to 10 μM of iEHZ2 or dEZH2 for 24 hours. (F) The expression levels of NF-κB and PD-L1 proteins, which were compared to GAPDH, were detected by western blot analysis. Daudi cells were treated with 10 μM of iEHZ2 or dEZH2 for 48 hours. (G) Ramos and Daudi cells were plated with in vitro activated CD8+ T cells at an E:T ratio of 1:1. Co-cultured cells were treated with 10 nM iEZH2 or dEZH2. After 48 hours, PD-L1 levels were measured by flow cytometry. (H, I) Activated CD8+ T cells co-cultured (H) Ramos and (I) Daudi cells (E:T ratio of 5:1) were treated by 10 μg/mL aPD1 with 10 nM dEZH2 or iEHZ2 for 72 hours in the presence IFN-γ. Cell viability was determined by WST-8 assay. Error bars are shown as mean ± SD. Data analyzed using two-tailed unpaired student’s t-tests. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001.

Article Snippet: Nonspecific background was blocked with 10% bovine serum albumin (#BSAS0.1, Bovogen, VIC, AU) in PBS for 60 min. For removal lipofuscin autofluorescence in tissues, TrueBlack (#23007, Biotium, CA, USA) was treated for 30 sec. Each tissue was stained with primary antibodies against EZH2 (#5246, Cell Signaling Technology), c-MYC (#18583, Cell Signaling Technology) CD8 (#MA1-81692, Invitrogen), granzyme B (#17215, Cell Signaling Technology), IFN-γ (#AF-285-NA, Novus Biologicals, CO, USA), and cleaved caspase-3 (#9664, Cell Signaling Technology) overnight at 4°C.

Techniques: Inhibition, Expressing, Flow Cytometry, Control, Quantitative RT-PCR, Western Blot, In Vitro, Cell Culture, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Dual targeting of EZH2 and PD-L1 in Burkitt’s lymphoma enhances immune activation and induces apoptotic pathway

doi: 10.3389/fimmu.2025.1578665

Figure Lengend Snippet: Effect of dEZH2 and aPD1 treatment on Burkitt’s lymphoma growth and EZH2 expression. (A) Experimental timeline of the in vivo injection for combination therapy. (B) The liver of each treatment or control group was harvested, photographed at the end of treatment. (C) Plasma was collected at the end of treatment. IFN-γ concentration was measured by ELISA. (D) EZH2 (green) and c-MYC (red) expressions in the liver were indicated on the immunofluorescence images. Mean fluorescence intensity (MFI) of EZH2 and c-MYC were shown in the graph. Scale bar = 200 µm. Error bars are shown as mean ± SD. Data analyzed using two-tailed unpaired student’s t-tests. * P ≤ 0.05; ** P ≤ 0.01.

Article Snippet: Nonspecific background was blocked with 10% bovine serum albumin (#BSAS0.1, Bovogen, VIC, AU) in PBS for 60 min. For removal lipofuscin autofluorescence in tissues, TrueBlack (#23007, Biotium, CA, USA) was treated for 30 sec. Each tissue was stained with primary antibodies against EZH2 (#5246, Cell Signaling Technology), c-MYC (#18583, Cell Signaling Technology) CD8 (#MA1-81692, Invitrogen), granzyme B (#17215, Cell Signaling Technology), IFN-γ (#AF-285-NA, Novus Biologicals, CO, USA), and cleaved caspase-3 (#9664, Cell Signaling Technology) overnight at 4°C.

Techniques: Expressing, In Vivo, Injection, Control, Clinical Proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Fluorescence, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Dual targeting of EZH2 and PD-L1 in Burkitt’s lymphoma enhances immune activation and induces apoptotic pathway

doi: 10.3389/fimmu.2025.1578665

Figure Lengend Snippet: The signaling pathway of anti-PD-1 and EZH2 degradation that induces apoptosis in Burkitt’s lymphoma. EZH2 degrader suppresses the expression of c-MYC, a key oncogene in Burkitt’s lymphoma. Also, this degradation promotes the transcriptional regulation of PD-L1 through NF-kB and IRF-1, which regulate PD-L1 expression on the tumor cell surface. The anti-PD-1 inhibits the PD-1/PD-L1 immune checkpoint pathway, effectively preventing the PD-L1 expressed on tumor cells from binding to PD-1 on the CD8+ T cell. This inhibition restores T cell activation, thereby enabling the release of cytotoxic molecules, such as IFN-γ and granzyme B. The increased level of granzyme B and EZH2 degradation activates apoptotic pathways, particularly by promoting the activation of caspase-3. Cleavage of caspase-3 and PARP signifies the induction of apoptosis. The combination of anti-PD-1 and EZH2 degradation amplifies the immune response while directly targeting apoptotic pathways, effectively inducing apoptosis in Burkitt’s lymphoma.

Article Snippet: Nonspecific background was blocked with 10% bovine serum albumin (#BSAS0.1, Bovogen, VIC, AU) in PBS for 60 min. For removal lipofuscin autofluorescence in tissues, TrueBlack (#23007, Biotium, CA, USA) was treated for 30 sec. Each tissue was stained with primary antibodies against EZH2 (#5246, Cell Signaling Technology), c-MYC (#18583, Cell Signaling Technology) CD8 (#MA1-81692, Invitrogen), granzyme B (#17215, Cell Signaling Technology), IFN-γ (#AF-285-NA, Novus Biologicals, CO, USA), and cleaved caspase-3 (#9664, Cell Signaling Technology) overnight at 4°C.

Techniques: Expressing, Binding Assay, Inhibition, Activation Assay

Journal: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A-B ) Representative confocal fluorescence microscopy images of endogenous EZH2 (A) or SUZ12 (B) immunostaining in MDA-MB-231 and BoM-1833 cells. Insets highlight exemplary nuclear bodies of EZH2 or SUZ12 accumulation (arrows) in the BoM-1833 cells. Scale bar: 10 µm. Images were acquired and are displayed with identical settings. ( C ) Violin plot quantifying PRC2 body diameter in BoM-1833 cells. Each dot represents a single PRC2 body; data from 3 biological replicates (N = 16–32 cells). ( D ) Quantification of percentage of cell nuclei with PRC2 bodies in MDA-MB-231 and BoM-1833 cells, based on the images representatively shown in A-B. Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, p=0.0102. Error bars indicate mean ±SEM. ( E ) Representative confocal fluorescence microscopy image of BoM-833 cells stained for endogenous PRC2 (SUZ12, green) and H3K27me3 (magenta) immunostaining in BoM-1833 cells. The arrow indicates an exemplary area of co-localization at a PRC2 body. Scale bar: 5 µm. ( F ) Schematic representation of the 3D photo-biotinylation approach used to map the proteome of endogenous PRC2 bodies. Total EZH2 (green) is spatially distributed within the cell and selectively photo-biotinylated at defined regions of interest (magenta) upon light activation. Following cell lysis, biotinylated proteins are captured using avidin-based immunoprecipitation and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The figure was created using Biorender. ( G ) Volcano plot illustrating the proteomic content of PRC2 bodies in BoM-1833 cells. Analysis was performed on the 1384 proteins identified as enriched in the labeled versus control condition in all 4 biological repeats, with unique peptides ≥ 2, fold change ≥ 1.5; and t-test significance ≤ 0.05. The x-axis represents the log 2 enrichment ratio (2P/CTL), and the y-axis represents the -log 10 p-value, indicating statistical significance. The dotted horizontal line corresponds to the p-value threshold (p < 0.05). Members of the core PRC2 complex are labeled in green. ( H ) Representative confocal fluorescence microscopy images of endogenous PHF19 immunostaining in MDA-MB-231 and BoM-1833 cells. The arrow highlights exemplary accumulations of PHF19 within nuclear bodies in BoM-1833 cells. Scale bar: 20 µm. The images were acquired and are displayed with identical settings. ( I ) Violin plot showing the quantification of endogenous PHF19 body diameter in BoM-1833 cells based on the images representatively shown in (H). Data represent measurements from N = 14–17 cells across n = 3 biological replicates, with each dot representing the diameter of a single PHF19 body. Biological repeats are color coded. ( J ) Quantification of percentage of cell nuclei with PHF19 bodies in MDA-MB-231 and BoM-1833 cells, based on the images representatively shown in (I). Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, p=0.003. Error bars indicate mean ±SEM. ( K ) Representative confocal fluorescence microscopy image of endogenous PHF19 (green) and H3K27me3 (magenta) immunostaining in BoM-1833 cells. The arrow indicates an exemplary area of co-localization at a PHF19 body. Scale bar: 5 µm. ( L ) Representative confocal fluorescence microscopy images of BoM-1833 cells, 24 h post transfection with a GFP-PHF19 (green) expression plasmid and immunostained for endogenous core PRC2 subunits (SUZ12, purple). The arrow indicates an exemplary area of co-localization. Scale bar: 10 µm.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Fluorescence, Microscopy, Immunostaining, Staining, Activation Assay, Lysis, Avidin-Biotin Assay, Immunoprecipitation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Labeling, Control, Transfection, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A-B ) Representative confocal fluorescence microscopy images of BoM-1833 cells transfected with the indicated siRNAs. Cells were fixed 96 hours post-transfection and immunostained for endogenous EZH2 (A) or SUZ12 (B). Regions of interest (ROIs) are highlighted, with inset images showing magnified views of the immunostained cells. Scale bar: 10 µm. Images that are to be directly compared where imaged and are displayed with identical settings. ( C ) Quantification of the percentage of nuclei exhibiting PRC2 bodies in BoM-1833 cells treated as in (A-B) and immunostained for PRC2 core subunits. Data represent measurements from N = 50–60 cells across n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA testing, *** = 0.0003, ns= not significant. Error bars indicate mean ±SD. ( D ) BoM-1833 cells were transfected with the indicated siRNAs and lysed 96 hours later for Western blot analysis using the specified antibodies. GAPDH was used as loading control. ( E-I ) Densitometric analysis of PHF19 (E), EZH2 (F), SUZ12 (G), PHF1 (H) and MTF2 (I) protein levels in cell lysates obtained from BoM-1833 cells treated as described in (D). GAPDH was used for relative normalization of the chemiluminescence signal obtained for the different PRC2 subunits. Data represent measurements from n = 3 biological replicates, whereby the values for siPHF19 are reported relative to the mean value of the control (siNT) within each biological replicate. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA testing, **** < 0.0001, ns = not significant. Error bars indicate mean ±SD.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Fluorescence, Microscopy, Transfection, Western Blot, Control

Journal: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A ) PHF19 gene expression analysis across a TCGA BRCA cohort sorted by molecular subtype subtype. Box plots display the expression levels of PHF19 in normal (grey) and tumor (green) tissue for the indicated breast cancer subtypes. Data are derived from TCGA/GTEx datasets and visualized using GEPIA2. Statistical significance between tumor and normal samples was determined by unpaired t-test (*p < 0.05). n= 291 (Normal), 194 (Luminal B), 415 (Luminal A), 66 (HER2), 135 (Basal-like). ( B-C ) Representative confocal microscopy images of EZH2 (B) and SUZ12 (C) immunostaining in the indicated cell lines. Scale bar: 20 µm. Images that are to be directly compared were recorded and are displayed using identical settings. ( D ) Quantification of the percentage of cell nuclei with PRC2 bodies in the indicated cell lines based on confocal microscopy images as shown in (B-C). Data represent measurements from N = 35– 55 cells across n = 3 biological replicates. Biological repeats are color coded. ( E ) Representative immunoblot analysis of full cell lysates prepared from the indicated cell lines and using the annotated antibodies. GAPDH was used as the loading control. ( F-G ) Densitometric quantification of EZH2, SUZ12 (F) and PCL family (G) subunit protein expression in the TNBC cell line panel used in this work. GAPDH was used for normalization of the chemiluminescence signal of the PRC2 subunits across cell lines. The data for siPHF19 are reported relative to the mean values for the siNT control. Data represent measurements from n = 3 biological replicates, error bars are mean ±SD. Measurements stemming from cell lines forming detectable PRC2 bodies by Airyscan microscopy were highlighted in red. ( H-I ) Representative confocal fluorescence microscopy images showing co-immunostaining of H3K27me3 with the endogenous PRC2 core subunit SUZ12 (H) and PHF19 (I) in MDA-MB-436 cells. Arrows indicate exemplary regions of colocalization. Scale bar: 10 µm (H), 5 µm (I). ( J ) Violin plot showing the quantification of PRC2 core and PHF19 protein body diameter as based on the images representatively shown in (F-G). Data represent measurements from N = 14–29 (core PRC2 subunits) and N= 19-22 (PHF19) cells across n = 3 biological replicates, with each dot representing the diameter of a single protein body. Biological repeats are color coded. ( K ) Representative confocal fluorescence microscopy images of MDA-MB-436 cells, 24 h post transfection with GFP-PHF19 (green) and immunostained for endogenous SUZ12 (purple). The arrow indicates an exemplary area of co-localization. Scale bar: 5 µm. ( L-M ) MDA-MB-436 cells were transfected with the indicated siRNAs followed by fixation 96 h later and immunostaining for endogenous EZH2 (L) or SUZ12 (M). The bottom row shows magnified views of the cropped fields of view. Images that are to be directly compared were acquired and are displayed using identical settings. Scale bar: 10 µm ( N ) Quantification of percentage of cell nuclei with PRC2 bodies in MDA-MB-436 cells transfected with the indicated siRNAs and imaged as representatively shown in (L-M). Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA, ****= 0.001, ns= not significant. Error bars indicate mean ±SD. ( O ) MDA-MB-436 were treated as described in (L-M), followed by cell lysis. The material was analyzed by Western blot using the indicated antibodies. See also Figure S4. ( P , S ) Representative confocal microscopy images and ( R , T ) quantification of HS578T (P, R) and BT549 (S, T) fixed 24 h after transfection with a plasmid encoding for GFP-PHF19 (magenta) and immunostained for endogenous SUZ12 (PRC2 core). ROIs (Regions of Interest) are highlighted and magnified, showing the endogenous localization of SUZ12 in cells transfected with GFP-PHF19 (ROI 1) versus un-transfected cells (ROI 2). Scale bar: 20 µm. The bar diagrams show the endogenous SUZ12 localization phenotype in relation to the GFP-PHF19 expression status. Data represent measurements from N = 7–30 cells from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, * = 0.0123, **= 0.0038. Error bars indicate mean ±SD.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Gene Expression, Expressing, Derivative Assay, Confocal Microscopy, Immunostaining, Western Blot, Control, Microscopy, Fluorescence, Transfection, Lysis, Plasmid Preparation