Journal: bioRxiv

Article Title: Active EB1 surges promote tubulin influx into the growing outer segments of the bipartite olfactory cilia in Drosophila

doi: 10.1101/2024.09.10.612170

Figure Lengend Snippet: A) Copurification of EB1:GFP from the chaGal4>EB1:GFP expressing Drosophila head extracts with the recombinant Glutathione S-transferase (GST)-tagged, KLP68D tail (GST-KLP68DT) fragment using affinity chromatography. The arrow indicates the EB1:GFP band. B) The immune-coprecipitation (IP) of the recombinant KLP68D from the head extracts of chaGal4>Klp68D:YFP and chaGal4>Klp68D(ΔT)YFP, expressing UAS-EB1FLAG using anti-FLAG. The arrow indicates a full-length KLP68DYFP band.

Article Snippet: Proteins from these gels were transferred onto a previously activated PVDF membrane (Hybond-P, GE Healthcare Ltd) in an electro-blotting apparatus (Bio-Rad, USA) following the supplier’s protocol and incubated in different primary antisera solutions as the following: anti-GFP Rabbit (dilution, 1:500; #3999 Bio Vision Inc., CA, USA) or anti-FLAG Rabbit (1:500; #F7425 Sigma-Aldrich, USA) Or anti-GST mouse (1:1000, Bioklone Biotech Pvt.

Techniques: Copurification, Expressing, Recombinant, Affinity Chromatography

Journal: bioRxiv

Article Title: N-Cadherin Provides a Cis and Trans Ligand for Astrotactin that Functions in Glial-Guided Neuronal Migration

doi: 10.1101/357541

Figure Lengend Snippet: Flow cytometry of live HEK 293T cells labeled with GFP and Alexa Fluor 647 antibodies. Control, untransfected cells (A) or cells transfected with Venus (B) , Astnl-FL-Venus (C) , or Astnl-ΔCTD-Venus , lacking the MACPF, FNIII and ANX-like domains in the C-terminus (D) . The x-axis shows total GFP fluorescence and the y-axis shows surface labeling (Alexa Fluor 647). Thus, cells expressing cytosolic Venus-tagged proteins are indicated in the lower right quadrant (Q3), while double positive cells (GFP+/Alexa Fluor 647+) in the upper right quadrant (Q2) express Venus-tagged proteins exposed on the cell surface. The percentage of cells expressing ASTN1 on the cell surface is depicted. Both ASTN1-FL and ASTNi-ΔCTD were significantly expressed on the cell surface (58 % and 49 % of live transfected cells, respectively).

Article Snippet: After removing the beads, the lysates were incubated with 3 μg of a rabbit GFP antibody (Invitrogen), rabbit Astn1 antibody, or normal rabbit IgG (Santa Cruz Biotechnology) for 2 h at 4 o C. Immunoprecipitates were collected on 50 μl Protein G/A Agarose beads by overnight rotation at 4 o C, washed with lysis buffer and resuspended in 50 μl 2X Laemmli buffer.

Techniques: Flow Cytometry, Labeling, Transfection, Fluorescence, Expressing

Journal: bioRxiv

Article Title: N-Cadherin Provides a Cis and Trans Ligand for Astrotactin that Functions in Glial-Guided Neuronal Migration

doi: 10.1101/357541

Figure Lengend Snippet: Drosophila S2 cell adhesion assays were prepared in four conditions: Cdh2;GFP + mCherry;GFP (A) , Cdh2;GFP + Astn1;mCherry (B) , Cdh2;GFP + Astn1;mCherry + ASTN1 Fab (C) , and Cdh2-A390;GFP + Astn1;mCherry (D) . ASTN1-positive cells were adhering to the CDH2-expressing aggregates after 30 min (arrows in B1 ), with more co-aggregation seen after 1 h (arrows in B2 ) and 2 h (arrows in B3 ), indicating heterophilic trans interactions. Significantly lower proportions of cells were adhering to the aggregates in the conditions with cells expressing control vector (A) or Astn1;mCherry blocked with ASTN1 Fab fragments (C) . The proportion of mCherry expressing cells in the CDH2;GFP-positive aggregates is quantified in (E) . Expression of CDH2∆390;GFP did not result in cell aggregation within 2 h (D) , demonstrating the importance of the cadherin ectodomain for homophilic and heterophilic interactions and cell adhesion. ** P < 0.01; *** P < 0.001. Scale bar represents 20 μm in (A - D) and 10 μm in inset in (B3).

Article Snippet: After removing the beads, the lysates were incubated with 3 μg of a rabbit GFP antibody (Invitrogen), rabbit Astn1 antibody, or normal rabbit IgG (Santa Cruz Biotechnology) for 2 h at 4 o C. Immunoprecipitates were collected on 50 μl Protein G/A Agarose beads by overnight rotation at 4 o C, washed with lysis buffer and resuspended in 50 μl 2X Laemmli buffer.

Techniques: Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: ZNF143 is a transcriptional regulator of nuclear-encoded mitochondrial genes that acts independently of looping and CTCF

doi: 10.1101/2024.03.08.583864

Figure Lengend Snippet: (A) Relative contact probability plot (top panel) and its derivative (bottom panel) calculated from the Hi-C matrices of DMSO (blue) and 6 hours dTAG-V1 (orange) treated ZFP143-FKBP cells. (B) Average cohesin (left), enhancer-promoter (E-P, middle) and promoter-promoter (P-P, right) loops in DMSO and 6 hours dTAG-V1 treated ZFP143-FKBP cells. Value in the upper-right corner indicates the interaction strength of the loop over the background. (C) Same as in (B) but for the average ZFP143-associated loops (containing ZFP143 peak in at least one loop anchor). (D) High-resolution 4C-seq data generated for the ZFP143-bound genes Rbm41 (left panel) and Prmt6 (middle panel), and non-ZFP143-bound control gene Sik1 (right panel), using gene promoters as viewpoints. The matrix in the top panel represents interaction frequencies in a previously published high-resolution Micro-C dataset . The arrows point to detected Micro-C chromatin loops. The bottom panel shows 4C contact profiles in DMSO (blue) and in 6 hours dTAG-V1 (orange) treated ZFP143-FKBP cells. Genomic tracks show ZFP143-HA ChIP-seq (red), calibrated CTCF ChIP-seq (blue), TT-seq nascent transcription (yellow for sense and purple for antisense transcription) in control and 6 hours dTAG-V1 treated ZFP143-FKBP cells. (E) Tornado plots of ZFP143-HA ChIP-seq signal centred at CTCF peaks in DMSO and 6 hours dTAG-V1 treated ZFP143-FKBP cells. (F) Same as in (E) but for the calibrated CTCF ChIP-seq signal centred at ZFP143-HA peaks.

Article Snippet: After systematically re-analysing the ZNF143 ChIP-seq data, we posit that the Proteintech anti-ZNF143 polyclonal antibody recognises CTCF in addition to ZNF143.

Techniques: Hi-C, Generated, Control, ChIP-sequencing

Journal: bioRxiv

Article Title: ZNF143 is a transcriptional regulator of nuclear-encoded mitochondrial genes that acts independently of looping and CTCF

doi: 10.1101/2024.03.08.583864

Figure Lengend Snippet: (A) Average Hi-C loops in DMSO and 6 hours dTAG-V1 treated ZFP143-FKBP cells. Value in the upper-right corner indicates the interaction strength of the loop over the background. (B) Same as in (A) but for the average ZFP143-associated Hi-C loops (containing ZFP143 peak in at least one loop anchor). (C) High-resolution 4C-seq data generated for the Cpox and Cldn1 (left panel) and Zfp111 and Zfp108 (right panel) loci using gene promoters as viewpoints. The matrix in the top panel represents interaction frequencies in a previously published high-resolution Micro-C dataset . The arrows point to detected Micro-C chromatin loops. The bottom panel shows 4C contact profiles in DMSO (blue) and in 6 hours dTAG-V1 (orange) treated ZFP143-FKBP cells. Genomic tracks show ZFP143-HA ChIP-seq (red), calibrated CTCF ChIP-seq (blue), TT-seq nascent transcription (yellow for sense and purple for antisense transcription) in DMSO and 6 hours dTAG-V1 treated ZFP143-FKBP cells. (D) Tornado plots of calibrated CTCF ChIP-seq signal centred at CTCF peaks in DMSO and 6 hours dTAG-V1 treated ZFP143-FKBP cells. (E) Genomic tracks showing ZFP143-HA ChIP-seq (red) in DMSO and calibrated CTCF ChIP-seq (blue) in DMSO and 6 hours dTAG-V1 treated ZFP143-FKBP cells. (F) Venn diagram showing the overlap between ZFP143-HA (red) and CTCF (blue) peaks.

Article Snippet: After systematically re-analysing the ZNF143 ChIP-seq data, we posit that the Proteintech anti-ZNF143 polyclonal antibody recognises CTCF in addition to ZNF143.

Techniques: Hi-C, Generated, ChIP-sequencing

Journal: bioRxiv

Article Title: ZNF143 is a transcriptional regulator of nuclear-encoded mitochondrial genes that acts independently of looping and CTCF

doi: 10.1101/2024.03.08.583864

Figure Lengend Snippet: (A) Overlap between ZNF143/ZFP143 peaks from re-analysed publicly available data and CTCF peaks from CISTROME for human (left panel) and mouse (right panel) datasets. Box plots for each ZNF143/ZFP143 dataset represent the median overlap with CTCF peaks. Each dot represents the overlap between the indicated ZNF143/ZFP143 peak set with an individual CTCF peak set. Colours represent the antibody used for chromatin immunoprecipitation, as indicated below. (B) Venn diagram showing the overlap between ZNF143 peaks detected by Proteintech (light pink) and FLAG (light green) antibodies in K562 cells. (C) Heatmap showing the enrichment of SBS (i.e. ZNF143) and CTCF motifs in common, Proteintech-specific, and FLAG-specific peaks in K562 cells. (D) Tornado plots of ChIP-seq signals detected by Proteintech (light pink), FLAG (light green), and custom (orange) antibodies, and CTCF signal (blue) in K562 cells. The ChIP-seq signals are centred on common (top) and Proteintech-specific (bottom) peaks. (E) Genomic tracks showing ChIP-seq signals for CTCF (blue) and signals detected by Proteintech (pink), FLAG (light green), and custom12 (orange) antibodies in K562 cells. Rectangles indicate common (left) and Proteintech-specific (middle and right) peaks in the region. (F) Scatter plot of the percentage of loop anchors overlapping the peak (x-axis) against the fold enrichment of peaks in loop anchors (y-axis) for a number of DNA binding proteins and for Proteintech-specific, FLAG-specific and common peaks in K562 cells.

Article Snippet: After systematically re-analysing the ZNF143 ChIP-seq data, we posit that the Proteintech anti-ZNF143 polyclonal antibody recognises CTCF in addition to ZNF143.

Techniques: Chromatin Immunoprecipitation, ChIP-sequencing, DNA Binding Assay

Journal: bioRxiv

Article Title: ZNF143 is a transcriptional regulator of nuclear-encoded mitochondrial genes that acts independently of looping and CTCF

doi: 10.1101/2024.03.08.583864

Figure Lengend Snippet: (A) Tornado plots of CTCF ChIP-seq signal from two biological replicates in wild-type (WT) and ZNF143-knockout (KO) haematopoietic stem and progenitor cells (HSPC) centred at ZNF143-related (top) and ZNF143-unrelated (bottom) CTCF peaks . (B) Same as in (A) but for the CTCF ChIP-seq signal in HSPC from two orthogonal studies , . (C) Rolling mean of the normalised CTCF motifs scores, annotated for the ZNF143-related (top) and ZNF143-unrelated (bottom) CTCF peaks. (D) Violin plots showing the fraction of ZNF143-related (left) and ZNF143-unrelated (right) CTCF peaks overlapping CTCF peaks from the CISTROME database . (E) GC bias scores calculated for CTCF ChIP-seq data generated from WT and ZNF143-KO HSPC samples . Note the divergence of the first WT CTCF replicate from the rest of the samples. (F) Genomic tracks showing CTCF ChIP-seq signal from two biological replicates in WT and ZNF143-KO HSPC , CTCF ChIP-seq signal from two other HSPC samples , , and GC content. Horizontal bars indicate ZNF143-related and ZNF143-unrelated CTCF peaks . Note the overlap of ZNF143-related peaks with GC-rich regions. (G) Tornado plots of ZNF143 ChIP-nexus signal from control and CTCF-depleted HEC1B cells centred at ZNF143-only (top) and shared ZNF143 and CTCF (bottom) peaks . (H) Genomic tracks showing ZNF143 ChIP-nexus signal from control and CTCF-depleted HEC1B cells . Horizontal bars indicate ZNF143-only and shared ZNF143 and CTCF peaks. Note the specific loss of signal at shared peaks upon CTCF depletion. (I) Venn diagram showing the overlap between ZNF143-CTCF motif pairs located 37 bp apart from each other and SINE/B2 repeat elements in the mouse genome from RepeatMasker. (J) Tornado plots of CTCF and ZNF143 ChIP-seq signal centred at ZNF143-CTCF motif pairs located 37 bp apart from each other .

Article Snippet: After systematically re-analysing the ZNF143 ChIP-seq data, we posit that the Proteintech anti-ZNF143 polyclonal antibody recognises CTCF in addition to ZNF143.

Techniques: ChIP-sequencing, Knock-Out, Generated, Control

Journal: bioRxiv

Article Title: Drosophila melanogaster Toll-9 elicits antiviral immunity against Drosophila C virus

doi: 10.1101/2024.06.19.599730

Figure Lengend Snippet: (A) In silico prediction of signal peptide in Toll-9 protein sequence. Red solid line indicates predicted n-terminal region, orange solid line indicates the predicted center hydrophobic region, and yellow solid line indicates predicted c-terminal region of signal peptide. Black dotted line indicates the cleavage site (CS) of the signal peptide. Sec/SPI: Sec translocon transported secretory signal peptide/Signal Peptidase I Tat/SPI: Tat translocon transported Tat signal peptides/Signal Peptidase I (B) Western blot analysis demonstrating the presence of Toll-9/V5 in endosomes. Endosomal fractions were identified using Rab5 as a microsomal marker, while Actin served as a cytosolic marker. Data are representative of three independent experiments. (C) Micrographs show colocalization of Rab5-early endosome marker (green) and Toll-9 (anti-V5 tag ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (D) Micrographs show colocalization of Rab7-Late endosome marker (green) and Toll-9 (anti-V5 tag ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (E) Micrographs show colocalization of Rab5-early endosome marker (green) and Poly(I:C) (J2 anti-dsRNA ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (F) Toll-9 (anti-V5 tag ab-Green) and Poly(I:C) (J2 anti-dsRNA ab-Red) in Poly(I:C) and CuSO 4 (500 µM) treated Toll-9 OE and S2 cells. (G) Western blot analysis using the indicated antibodies following immunoprecipitation of V5 tag (Toll-9) using J2 dsRNA antibody from the lysate of Poly (I:C) treated Toll-9 OE and S2 cells in presence and absence of CuSO 4 (500 µM). Data are representative from three independent experiments.

Article Snippet: The cells were blocked in phosphate-buffered saline (PBS) containing 10% FBS and incubated with antibodies against Rab5 (1:50; Abcam ab31261), Rab7(1:20; Developmental Studies Hybridoma Bank Rab7), DCV Capsid (1:200; Abcam ab92954), dsRNA J2 (1:200; Jena Biosciences RNT-SCI-10010200) and V5 tag (1:200; ThermoFisher Scientific R960-25) for overnight at 4°C.

Techniques: In Silico, Sequencing, Western Blot, Marker, Immunoprecipitation

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

Journal: bioRxiv

Article Title: Ellagic acid: a potential inhibitor of enhancer of zeste homolog-2 and protein arginine methyltransferase-5

doi: 10.1101/2024.05.22.595443

Figure Lengend Snippet: Following exposure of MDA-MB231 cells to EA for 24 hours, it was noted that EA induced autophagy in these cells. (a) Treatment with EA resulted in the formation of acidic vesicular organelles (AVOs) in cells. (b & c) Western blot analysis was performed to notice the expression of LC3 I/II and to evaluate the expression levels of Beclin-1, Bax, Bad and BCl2 proteins in MDA-MB231 cells treated upon with EA.

Article Snippet: The antibodies for signaling studies BCL-2 (A0208), Bax (A0207), Bad (A1593), Beclin-1 (A7353), LC3 I/II (A19665), obtained from Abclonal Biotech Company US.

Techniques: Western Blot, Expressing