Journal: bioRxiv

Article Title: Impact of histone deacetylase inhibition and arimoclomol on heat shock protein expression and disease biomarkers in primary culture models of familial ALS

doi: 10.1101/2023.12.13.571549

Figure Lengend Snippet: Effects of HDAC inhibitors, arimoclomol and combined treatments on HSPA1A in motor neurons expressing TDP-43 G348C . A Phase contrast image of spinal cord – DRG culture. Arrows point to motor neurons. B Small but significant increase in the percentage of motor neurons with HSPA1A immunoreactivity three days following microinjection of plasmid encoding TDP-43 G348C , compared to absence of labeling in neurons injected with mCherry (control). C-F Percentage of HSPA1A immunopositive motor neurons after three days of treatment with vehicle, the HDAC inhibitors C SAHA, D RGFP109, E RGFP963 or F Tubastatin A, or with arimoclomol alone or in combination with the respective HDAC inhibitor. HDAC inhibitors with class I activity ( C-E ) significantly increased the percentage of neurons expressing HSPA1A. Arimoclomol and the HDAC6 inhibitor Tubastatin A were ineffective. Data presented as mean ± S.D., n = 9 cultures. Statistical significance was evaluated by one-way ANOVA followed by Bonferroni post hoc analysis. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Scale bar = 20μm.

Article Snippet: Primary antibodies were: rabbit anti-TDP-43 (EPR18554 1:500 Abcam, Cambridge, UK), rabbit anti-FUS (11570-1-AP 1:300, Proteintech, Rosemont, IL, USA); mouse antibody specific for human SOD1 (SD-G6 1:100, Millipore Sigma), Mouse anti-human HSP70 specific for stress-inducible HSPA1A (SMC-100B 1:100, Stressmarq Biosciences Inc., Victoria, BC, Canada), mouse anti-HSPA8/HSC71/Hsc70 (13D3) antibody (NB120-2788 1:300, Novus Biologicals, Colorado, USA), mouse anti-FLAG M2 (F1804 1:400, Millipore Sigma), rabbit anti-acetylated H3K9/K14 (9677 1:400, Cell Signaling, Danvers, MA, USA), rabbit anti-Brg1 (21634-1-AP 1:600, Proteintech), rabbit anti-MAP2 (AB5622 1:500, Millipore Sigma), chicken anti-GFP (GFP1010 1:500 AVES, CA, USA).

Techniques: Expressing, Microinjection, Plasmid Preparation, Labeling, Injection, Control, Activity Assay

Journal: bioRxiv

Article Title: Impact of histone deacetylase inhibition and arimoclomol on heat shock protein expression and disease biomarkers in primary culture models of familial ALS

doi: 10.1101/2023.12.13.571549

Figure Lengend Snippet: Hspa1a mRNA in ALS culture models using single molecule fluorescence in situ hybridization (smFISH). Cultures containing motor neurons expressing TDP-43 G348C , FUS R521G or SOD1 G93A were treated with vehicle (DMSO), 4 µM arimoclomol, 1µM RGFP963 or the combination of arimoclomol and RGFP963 (Combo). smFISH was conducted on day three. A The highly expressed Hspa8 mRNA is presented as a reference control for comparison to the minimal labeling of Hspa1a mRNA in B,C,D . B No significant impact of TDP-43 G348C or drug treatments on the number of Hspa1a mRNA spots. C Scarcity of Hspa1a mRNA spots in neurons expressing FUS R521G ; no significant effect of drug treatments. D Low numbers of Hspa1a mRNA spots in neurons expressing SOD1 G93A ; a small but significant increase in a subset of neurons by arimoclomol and RGFP963 treatments. Data are presented as mean ± S.D. n = 9-24 neurons per group. E-G Number of transcription sites by smFISH. E Example of H spa1a transcription site labeling. F,G Effect of ALS variants and combination drug treatment on the number of Hspa1a transcription sites on F day one and G day three following microinjection of expression vectors. n = 6-30 neurons per group. Statistical significance was evaluated through one-way ANOVA followed by Bonferroni post hoc analysis. *p<0.05. Scale bar = 15μm.

Article Snippet: Primary antibodies were: rabbit anti-TDP-43 (EPR18554 1:500 Abcam, Cambridge, UK), rabbit anti-FUS (11570-1-AP 1:300, Proteintech, Rosemont, IL, USA); mouse antibody specific for human SOD1 (SD-G6 1:100, Millipore Sigma), Mouse anti-human HSP70 specific for stress-inducible HSPA1A (SMC-100B 1:100, Stressmarq Biosciences Inc., Victoria, BC, Canada), mouse anti-HSPA8/HSC71/Hsc70 (13D3) antibody (NB120-2788 1:300, Novus Biologicals, Colorado, USA), mouse anti-FLAG M2 (F1804 1:400, Millipore Sigma), rabbit anti-acetylated H3K9/K14 (9677 1:400, Cell Signaling, Danvers, MA, USA), rabbit anti-Brg1 (21634-1-AP 1:600, Proteintech), rabbit anti-MAP2 (AB5622 1:500, Millipore Sigma), chicken anti-GFP (GFP1010 1:500 AVES, CA, USA).

Techniques: Fluorescence, In Situ Hybridization, Expressing, Control, Comparison, Labeling, Microinjection

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Association between gene expression and CES total editing rate We analyzed association between CES total editing rate and gene expression for 14,961 human genes. (a) Gene set enrichment analysis by hypergeometric test on GO-BP categories and REACTOME pathways revealed that associated genes are mainly involved in immune system response mediated by interferon I and alpha / beta. (b) When we analyze distribution of CES total editing rate and ADAR gene expression, ADAR expression levels explains ∼ 13% of observed variability. No significant effect is observed for ADARB1 expression. ADAR and ADARB1 expression levels are reported as residuals of ridge regression with technical covariates (see description of data in methods section). The graphs report adjusted p-value and R2 value from robust regression analysis. (c) The 1,122 genes associated to CES total editing rate after removing ADAR expression effect were enriched for genes mainly involved in ribonucleoprotein and RNA processing.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Genes associated with CES total editing rate are enriched for ADAR interactors (a) Reconstructed PPI network including ADARs and proteins encoded by best genes significantly associated with global editing levels (FDR < 0.01). Among these proteins, we observed 285 potential ADARs interactors, including 9 direct partners of ADARs proteins. (b) Boxplot of number of ADARs interacting genes observed in 1M random simulations. The observed number of interactions (285) resulted in empirical p-value < 1e-6. (c) ADARs interactors are strongly enriched for RNA binding proteins in GO-MF categories. (d) Distribution of degree and betweenness centrality values among network nodes are represented by violin plots. ADAR1 protein has a major role (higher values) among ADAR proteins. Among ADARs direct partners, ELAVL1, RPA1 and IFI16 showed high values of degree and betweenness centrality, suggesting a central role in the network. (e) ADAR1 interaction with RPA70 (coded by RPA1) and IFI16 determined by co-immunoprecipitation. After immunoprecipitation with ADAR1 antibody, western blot for IFI16 and RPA70 are reported. For a better discrimination two times of exposure are reported in the figure.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: RNA Binding Assay, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Impact of cell composition on CES total editing rate and ADAR / ADARB1 expression Our analysis revealed strong associations with CES total editing rate for 4 cell type variables (a), representing proportion of neutrophils, monocytes, dendritic cells (DC) and T helper (Th). Specific cell variables resulted significantly associated also to ADAR (b) and ADARB1 (c) expression. Significance level (p) and correlation coefficient (r) are reported in each plot based on Pearson’s product-moment. Only non-zero observations are plotted.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Impact of biological / pharmacological factors on CES total editing rate and ADAR / ADARB1 expression Our analysis revealed significant associations with CES total editing rate for blood pressure medication, BMI current, Age and Sex (a). Specific biological variables resulted significantly associated also to ADAR (b) and ADARB1 (c) expression. Significance level of association after correction for cell composition is reported (p (cell)) is reported in each plot based on Mann-Whitney-Wilcoxon or Pearson’s product-moment correlation test for binary and continuous variables, respectively. For continuous variables the Pearson correlation coefficient (r) is also reported.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Impact of cell composition, biological and pharmacological factors on PCs of editing levels The heathmap represents strength of association between the first 5 principal components of CESs (PCs) and ADAR / ADARB1 expression (upper panel), 7 cell composition variables (middle panel) and 11 biological / pharmacological variables (lower panel). Only factors showing significant association with at least one of the first 5 PCs are represented. Significant p values (< 0.05) are colored in yellow-red scale, while p value > 0.05 are represented in grey scale. Age, BMI, blood pressure medications, smoke and alcohol all associate with PC1. Also time of blood draw seems to have a small, but consistent effect, on different PCs. For each PC, variance explained is represented by the bar plot in the upper side.

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing

Journal: bioRxiv

Article Title: Genome-wide analysis of consistently RNA edited sites in human blood reveals interactions with mRNA processing genes and suggests correlations with cell types and biological variables

doi: 10.1101/254045

Figure Lengend Snippet: Association study for SNPs and CES total editing rate (a) Manhattan plot representing the association between 573,801 SNPs and CES total editing rate, where black line represents threshold for the top 100 SNPs (p value ∼ 10e-4). (b) Detailed view of genotyped SNPs located in the region at chromosome 7 that showed significant association with CES total editing rate. Known GWAS associations for human phenotypes from GRASP database are reported in the lower panel. (c) The top associated SNP (rs856554) showed a significant effect on global editing level, while no significant correlation was observed with ADAR and ADARB1 expression. (d) Real-time expression analysis of ADAR and ADARB1 mRNA after B-EBV transfection of LOC730338. Not transfected cells were used as control samples. Data are reported as 2 -ΔΔ ct (expression level of control sample is equal to 1) and represent mean values and standard errors obtained from at least 3 independent evaluations. Unpaired t test was used for statistical analysis (*p< 0.05).

Article Snippet: During the immunoblot step the following primary antibodies were used 1h at RT: mouse anti-ADAR1 (Santa Cruz, cod. sc-73408) 1:300 in 5% non-fat dry milk in TBST 0,1%; mouse anti-IFI16 (Abcam, cod.

Techniques: Expressing, Transfection

Journal: bioRxiv

Article Title: The histone demethylase KDM5 is essential for larval growth in Drosophila

doi: 10.1101/297804

Figure Lengend Snippet: (A) Lethality of kdm5 K06801 , kdm5 10424 and kdm5 140 homozygous mutant animals generated from a cross between five female and five male heterozygous parents balanced using CyO-GFP. The column labeled total flies indicates the number of progeny (adult) flies scored from at least three independent crosses. Expected number of progeny is based on Mendelian frequencies and taking into account the lethality of CyO homozygotes, i.e 33% of total adult flies. * p <0.01 (chi-squared test). (B) Position of the NP4707 , 10424 and K06801 P element insertions and molecular mapping of the kdm5 140 deletion. Ab indicates the region used to generated the rabbit polyclonal anti-KDM5 antibody ( S ecombe et al . 2007 ). (C) RT-PCR using primers to the 5’ end of the gene using RNA from whole 3 rd instar larvae. Animals homozygous for kdm5 K06801 or kdm5 10424 show low levels of transcript while kdm5 140 shows none. kdm5 mRNA normalized to wildtype ( w 1118 ) using rp49 . **** p <0.0001. (D) RT-PCR using primers to the 3’ end of the gene using RNA from whole 3 rd instar larvae. kdm5 140 has wildtype levels of the 3’ end of the transcript. **** p <0.0001. ns = not significant. (E) Western from wildtype ( w 1118 ) and kdm5 140 homozygous mutant wing imaginal discs showing KDM5 and alpha tubulin. kdm5 140 animals have no detectable full length or truncated KDM5. *ns indicates non-specific band. (F) Schematic of strain genotype for rescue of kdm5 140 with a genomic rescue transgene. Flies are homozygous for the kdm5 140 mutation on the 2 nd chromosome and homozygous for an 11kb genomic rescue transgene on the 3 rd chromosome. (G) Western blot showing KDM5 protein levels from 3 rd instar larval wing imaginal discs from wildtype ( w 1118 ) and kdm5 140 homozygotes that also have two copies of the kdm5:HA genomic rescue transgene. Anti-KDM5 (top), anti-HA (middle) and anti-histone H3 loading control (bottom). (H) kdm5 140 lethality is rescued by a transgene encoding the kdm5 locus. These data were generated by crossing female and male flies heterozygous for kdm5 140 and homozygous the wildtype genomic rescue transgene (intercross of kdm5 140 /CyO-GFP; kdm5:HA / kdm5:HA males and females).

Article Snippet: Antibodies used were anti-pH3 (Cell signaling #9701, 1/1000), anti-histone H3 (Active Motif #39763 or #39163, 1/5000), anti-alpha Tubulin (Developmental Studies Hybridoma Bank, University of Iowa; 1:5000).

Techniques: Mutagenesis, Generated, Labeling, Reverse Transcription Polymerase Chain Reaction, 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-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