gsk  (MedChemExpress)

Journal: bioRxiv

Article Title: Polycomb Repressive Complexes 1 and 2 are recruited independently to pericentromeric heterochromatin in response to hypomethylation in mouse embryonic stem cells

doi: 10.1101/2025.11.14.688451

Figure Lengend Snippet: (A) Graphical representation of the macro used for unbiased quantification of signal within the chromocenters as explained in the methods. Briefly, the composite image is split into the individual channels. The DAPI channels is thresholded (Default method) to identify the area of the nucleus. The mean signal intensity is then measured in the DAPI channel and the bottom threshold for the chromocenters is calculated as 1.2 st.dev over the mean signal. The regions of the chromocenters are then subtracted from that of the nucleus to give the nucleoplasm. All regions are then added to ROI manager. The signal intensity in all objects in ROI manager are measured in the rest of the channels. The Mean signal intensity is then calculated as the Mean signal intensity in chromocenter divided by that in the nucleoplasm and multiplied by 100. (B) Violin plot showing the percentage of the chromocenter occupied by increased signal of the reporter/antibody in J1 and TKO cells. Significance was determined by Kruskal-Wallis (one-way Anova) test where **** p≤0.0001 (C) Violin plot of percentage of the Cbx7-GFP defined foci occupied by increased signal of the antibody (H2AK119ub and H3K9me3) in J1 and TKO cells. Significance was determined by Kruskal-Wallis (one-way Anova) test with FDR correction-Benjamini and Hochberg. Test was performed between J1 and TKO samples for each antibody. (D) Violin plot showing the percentage of the chromocenter occupied by increased signal of the reporter/antibody in Dnmt1 tet/tet cells with or without dox. Significance was determined by Kruskal-Wallis (one-way Anova) test where **** p≤0.0001 (E) Violin plot showing the chromocenter size (µm) in J1, TKO and Dnmt1 tet/tet cells with (4 days) or without dox. Significance was determined by ordinary one-way Anova test with FDR correction-Benjamini and Hochberg. Test was performed between every sample. Non-significant values are not shown. (F) Violin plot of chromocenter size (px) in Dnmt1 tet/tet cells in presence of dox for 4 days and 7 days. Significance was determined by ordinary one-way Anova test with FDR correction-Benjamini and Hochberg. Test was performed between every sample. (G) IF on WT and Dnmt TKO cells containing Cbx7-GFP reporter stained for H2AK119ub counterstained with DAPI. Representative mitotic nucleus from maximum intensity Z-projections from 3D stack are shown. C indicates position of centromere. Z-stacks obtained using 40x air objective. Scale bar corresponds to 5µm. (H) IF on Dnmt1 tet/tet cells containing Cbx7-GFP reporter stained for H2AK119ub counterstained with DAPI. Representative mitotic nuclei from maximum intensity Z-projections from 3D stack are shown C indicates position of centromere. Z-stacks obtained using 40x air objective. Scale bar corresponds to 5µm.

Article Snippet: Inhibition of DNMT1 protein in all other cell lines was performed using 2μM of the Dnmt1 inhibitor (MedChemExpress, #GSK-3484862) for 4 days unless otherwise stated.

Techniques: Staining

Journal: bioRxiv

Article Title: Polycomb Repressive Complexes 1 and 2 are recruited independently to pericentromeric heterochromatin in response to hypomethylation in mouse embryonic stem cells

doi: 10.1101/2025.11.14.688451

Figure Lengend Snippet: (A) Graphical representation of the Dnmt1 tet/tet cell line. Donated by Chaillet lab. Modified from Borowczyk et al., 2009 – lacking neomycin and puromycin resistance cassettes. (B) Western blot for Dnmt1 and H4 (loading control) proteins over 7 day dox treatment in for Dnmt1 tet/tet cells. M – Molecular ladder. Arrow points to expected band for Dnmt1 protein. (C) Southern blot using methylation sensitive restriction digest (HpyCH4IV enzyme –cut site shown below) followed by hybridisation using probe for minor satellite repeats in Dnmt1 tet/tet cells with (over 7 days) or without dox and in J1 and TKO cells. Methylated DNA cannot be digested and remains as high MW bands. Unmethylated DNA is digested and separates into polymers of satellite repeats. (D) HPLC Mass spectrometry analysis of global 5mC levels as percentage of total Guanines over 7 day dox treatment in for Dnmt1 tet/tet cells and TKO cells as control. Significance was determined by Kruskal-Wallis (one-way Anova) test with FDR correction – Benjamini, Krieger and Yekutieli. (E) Western blot for H3K27me3, H2AK119ub, H3K9me3 and H4 (loading control) over 7 day dox treatment in for Dnmt1 tet/tet cells. M – Molecular ladder. (F) Fixed immunofluorescence (IF) in Dnmt1 tet/tet cells over 7 days dox treatment and TKO cells stained for H2AK119ub (bottom row) and counterstained for DAPI (top row). Representative nuclei from single slice of 3D stack are shown. Z-stacks obtained using 40x air objective on an epifluorescent microscope. Scale bar corresponds to 5µm. Arrowheads point to a representative chromocenter at which the DAPI and antibody signals overlap. (G) Violin plot showing mean signal intensity of H2AK119ub antibody signal at chromocenter normalised to that in the nucleoplasm in Dnmt1 tet/tet cells over 7 days dox treatment and TKO cells. Grey dashed line corresponds the threshold above which the signal is considered increased at the chromocenter compared to the nucleoplasm. Significance was determined by Kruskal-Wallis (one-way Anova) test where **** p≤0.0001.

Article Snippet: Inhibition of DNMT1 protein in all other cell lines was performed using 2μM of the Dnmt1 inhibitor (MedChemExpress, #GSK-3484862) for 4 days unless otherwise stated.

Techniques: Modification, Western Blot, Control, Southern Blot, Methylation, Hybridization, Mass Spectrometry, Immunofluorescence, Staining, Microscopy

Journal: bioRxiv

Article Title: Polycomb Repressive Complexes 1 and 2 are recruited independently to pericentromeric heterochromatin in response to hypomethylation in mouse embryonic stem cells

doi: 10.1101/2025.11.14.688451

Figure Lengend Snippet: (A) Single timepoint snapshots from maximum intensity z-projection of Dnmt1 tet/tet cells without (top panel) or with dox for 4 days (bottom panel) with the Cbx7-GFP reporter (green signal), imaged over 2 hours. Scale bar corresponds to 20µm. (B) Live-cell imaging of Dnmt1 tet/tet cells without or with dox for 4 days containing the Cbx7-GFP reporter, imaged over 2 hours, image taken every 3 min. Representative nuclei at interphase (top two panels) and mitosis (bottom two panels) shown at indicated time points (every 15min). Scale bar corresponds to 10µm. Arrowheads point to clustering of Cbx7-GFP signal under dox conditions following exit from mitosis. (C) IF on Dnmt1 tet/tet cells containing Cbx7-GFP reporter stained for H2AK119ub counterstained with DAPI. Representative nuclei from single slice of 3D stack are shown. Z-stacks obtained using 40x air objective. Scale bar corresponds to 5µm. Arrowheads point to a representative chromocenter at which all three signals overlap. (D) IF for H2AK119ub counterstained with DAPI in Dnmt1 tet/tet cells in presence of dox and following dox recovery. Representative nuclei from single slice of 3D stack are shown. Z-stacks obtained using 40x air objective on an epifluorescent microscope. Scale bar corresponds to 5µm. Plot shows the signal intensity of each of the channels along the diagonal of the rectangle in each channel at the specific chromocenter (blue line - DAPI channel, green line - antibody signal. (E) Violin plot showing mean signal intensity of Cbx7-GFP reporter/H2AK119ub antibody signal at chromocenter normalised to that in the nucleoplasm in Dnmt1 tet/tet cells with or without dox. Grey dashed line corresponds the threshold above which the signal is considered increased at the chromocenter compared to the nucleoplasm. Significance was determined by Kruskal-Wallis (one-way Anova) test where **** p≤0.0001. Dark blue signifies high DNA methylation, Light blue corresponds to low DNA methylation. (F) Percentage of reads corresponding to satellite repeats to total number of reads from published H3K27me3 ChIP data on Dnmt1 tet/tet cells without, with and upon dox recovery and in J1 and TKO.

Article Snippet: Inhibition of DNMT1 protein in all other cell lines was performed using 2μM of the Dnmt1 inhibitor (MedChemExpress, #GSK-3484862) for 4 days unless otherwise stated.

Techniques: Live Cell Imaging, Staining, Microscopy, DNA Methylation Assay

Journal: bioRxiv

Article Title: Polycomb Repressive Complexes 1 and 2 are recruited independently to pericentromeric heterochromatin in response to hypomethylation in mouse embryonic stem cells

doi: 10.1101/2025.11.14.688451

Figure Lengend Snippet: (A) Schematic representation of the CRISPR strategy and sgRNA targeting to generate Ezh2 KO. Adapted from (Lavarone, Barbieri and Pasini, 2019). Primer pairs used for genotyping are shown. CRISPR guide PAM sites are shown to be within the exons. (B) Homozygosity PCR screen using two primer pairs in which one of the primers binds inside the deleted region. Successful Ezh2 KO clones are expected to produce no band while wild type alleles would yield 440bp band Exon 2 primer pair (top two panels) and 290bp band for Exon 20 primer pair (bottom two panels). Selected clone (C3) used in this study is marked with red asterisk * (C) Western blot for Dnmt1, Ezh2, H3K27me3 and H4 (loading control) in Dnmt1 tet/tet cells and Ezh2 KO cells with or without dox at different time points. (D) Western blot for H2AK119ub and H4 (loading control) in Dnmt1 tet/tet cells and Ezh2 KO cells with or without dox at different time points. (E) Mean satellite read counts per million (CPM) from RNA sequencing in Dnmt1 tet/tet cells and Ezh2 KO cells with or without dox. Significance was determined by Ordinary one-way Anova with Tukey’s multiple comparisons test, ** p<0.01, *** p<0.001, **** p<0.0001. (F) Western blot for H2AK119ub, H3K27me3, Dnmt1, H3K9me3 and H4 (loading control) in Ring1b-AID cells treated with DMSO, auxin, Dnmt1i or both auxin and Dnmt1i. (G) Southern blot using methylation sensitive restriction digest (HpyCH4IV enzyme –cut site shown below) followed by hybridisation using probe for minor satellite repeats in Ring1b-AID cells treated with DMSO, auxin, Dnmt1i or both auxin and Dnmt1i.

Article Snippet: Inhibition of DNMT1 protein in all other cell lines was performed using 2μM of the Dnmt1 inhibitor (MedChemExpress, #GSK-3484862) for 4 days unless otherwise stated.

Techniques: CRISPR, Clone Assay, Western Blot, Control, RNA Sequencing, Southern Blot, Methylation, Hybridization

Journal: bioRxiv

Article Title: Polycomb Repressive Complexes 1 and 2 are recruited independently to pericentromeric heterochromatin in response to hypomethylation in mouse embryonic stem cells

doi: 10.1101/2025.11.14.688451

Figure Lengend Snippet: (A) IF for H2AK119ub counterstained with DAPI in Dnmt1 tet/tet cells and Ezh2 KO cells with or without dox. Representative nuclei from single slice of 3D stack are shown. Z-stacks obtained using 40x air objective. Scale bar corresponds to 5µm. Arrowheads point to a representative chromocenter at which the DAPI and antibody signals overlap. (B) Violin plot of mean signal intensity of H2AK119ub antibody signal at chromocenter normalised to that in the nucleoplasm in Dnmt1 tet/tet cells and Ezh2 KO cells with or without dox. Grey dashed line corresponds the threshold above which the signal is considered increased at the chromocenter compared to the nucleoplasm. Significance was determined by Kruskal-Wallis (one-way Anova) test where **** p≤0.0001. (C) As in (A) IF for Cbx7-GFP reporter counterstained with DAPI in Ring1b-AID cells treated with DMSO, auxin, Dnmt1i or both auxin and Dnmt1i. Arrowheads point to a representative chromocenter at which the DAPI and reporter signals overlap. (D) As in (B) Violin plot of mean signal intensity of Cbx7-GFP reporter signal at chromocenter normalised to that in the nucleoplasm in Ring1b-AID cells treated with DMSO, auxin, Dnmt1i or both auxin and Dnmt1i.

Article Snippet: Inhibition of DNMT1 protein in all other cell lines was performed using 2μM of the Dnmt1 inhibitor (MedChemExpress, #GSK-3484862) for 4 days unless otherwise stated.

Techniques:

Journal: bioRxiv

Article Title: Polycomb Repressive Complexes 1 and 2 are recruited independently to pericentromeric heterochromatin in response to hypomethylation in mouse embryonic stem cells

doi: 10.1101/2025.11.14.688451

Figure Lengend Snippet: (A) Schematic representation of the CRISPR strategy and sgRNA targeting to generate Scml2 KO. Primer pairs used for genotyping are shown. CRISPR guide PAM sites are shown. (B) PCR screen for homozygous macrodeletion using two primer pairs in which one of the primers binds inside the deleted region. Successful deletion results in no band with the Exon 2 (top panel) and exon 26 (bottom panel) primer pairs. Selected clone (B4) is indicated by red asterisk *. (C) Plot showing normalised read counts of Scml2 gene in Dnmt1 tet/tet cells and Scml2 KO cells with or without dox. (D) Plot showing normalised read counts of Dnmt1 gene in Dnmt1 tet/tet cells and Scml2 KO cells with or without dox. (E) Plot showing normalised read counts of Dazl gene in Dnmt1 tet/tet cells and Scml2 KO cells with or without dox. (F) Mean satellite read counts per million (CPM) from RNA sequencing in Dnmt1 tet/tet cells and Scml2 KO cells with or without dox. Significance was determined by Ordinary one-way Anova with Tukey’s multiple comparisons test, ** p<0.01, *** p<0.001. (G) Graphical representation of the Scml2-GFP reporter containing the full Scml2 gene with two Malignant Brain Tumour (MBT) domains, 10 Scml2 DNA binding repeats (SBT), RNA binding region (RBR) domain and Scm-like embedded domain (SLED). EGFP domain was inserted at the C-terminus of the gene. (H) IF for H3K27me3 counterstained with DAPI in parental J1 and TKO as well as in the presence of ectopic Scml2-GFP. Representative nuclei from a max intensity Z-projection of 3D stack are shown. Z-stacks obtained using 60x air objective. Scale bar corresponds to 5µm.

Article Snippet: Inhibition of DNMT1 protein in all other cell lines was performed using 2μM of the Dnmt1 inhibitor (MedChemExpress, #GSK-3484862) for 4 days unless otherwise stated.

Techniques: CRISPR, RNA Sequencing, Binding Assay, RNA Binding Assay

Journal: bioRxiv

Article Title: Polycomb Repressive Complexes 1 and 2 are recruited independently to pericentromeric heterochromatin in response to hypomethylation in mouse embryonic stem cells

doi: 10.1101/2025.11.14.688451

Figure Lengend Snippet: (A) IF for Cbx7-GFP counterstained with DAPI in Dnmt1 tet/tet cells and Scml2 KO cells with or without dox. Representative nuclei from single slice of 3D stack are shown. Z-stacks obtained using 40x air objective. Scale bar corresponds to 5µm. Arrowheads point to a representative chromocenter at which the DAPI and reporter signals overlap. (B) Violin plot of mean signal intensity of Cbx7-GFP reporter signal at chromocenter normalised to that in the nucleoplasm in Dnmt1 tet/tet cells and Scml2 KO cells with or without dox. Grey dashed line corresponds the threshold above which the signal is considered increased at the chromocenter compared to the nucleoplasm. Significance was determined by Kruskal-Wallis (one-way Anova) test where **** p≤0.0001. Test was performed between every sample. Non-significant samples are not shown. (C) As in (A) IF for H2AK119ub counterstained with DAPI in Dnmt1 tet/tet cells and Scml2 KO cells with or without dox. Arrowheads point to a representative chromocenter at which the DAPI and antibody signals overlap. (D) As in (B) Violin plot of mean signal intensity of H2AK119ub antibody signal at chromocenter normalised to that in the nucleoplasm in Dnmt1 tet/tet cells and Scml2 KO cells with or without dox.

Article Snippet: Inhibition of DNMT1 protein in all other cell lines was performed using 2μM of the Dnmt1 inhibitor (MedChemExpress, #GSK-3484862) for 4 days unless otherwise stated.

Techniques:

Journal: bioRxiv

Article Title: Polycomb Repressive Complexes 1 and 2 are recruited independently to pericentromeric heterochromatin in response to hypomethylation in mouse embryonic stem cells

doi: 10.1101/2025.11.14.688451

Figure Lengend Snippet: (A) Schematic representation of the CRISPR strategy and sgRNA targeting to generate Bend3 KO. Primer pairs used for genotyping are shown. CRISPR guide PAM sites are shown. (B) PCR screen for homozygous macrodeletion using two primer pairs in which one of the primers binds inside the deleted region. Successful deletion results in no band with the Exon 2 (top panel) and exon 4 (bottom panel) primer pairs. Selected clone (D7) is indicated by red asterisk *. (C) Plot showing normalised read counts of Bend3 gene in Dnmt1 tet/tet cells and Bend3 KO cells with or without dox. (D) Plot showing normalised read counts of Dnmt1 gene in Dnmt1 tet/tet cells and Bend3 KO cells with or without dox. (E) Plot showing normalised read counts of Dazl gene in Dnmt1 tet/tet cells and Bend3 KO cells with or without dox. (F) Mean satellite read counts per million (CPM) from RNA sequencing in Dnmt1 tet/tet cells and Bend3 KO cells with or without dox. Significance was determined by Ordinary one-way Anova with Tukey’s multiple comparisons test, ** p<0.01, *** p<0.001, **** p<0.0001.

Article Snippet: Inhibition of DNMT1 protein in all other cell lines was performed using 2μM of the Dnmt1 inhibitor (MedChemExpress, #GSK-3484862) for 4 days unless otherwise stated.

Techniques: CRISPR, RNA Sequencing

Journal: bioRxiv

Article Title: Polycomb Repressive Complexes 1 and 2 are recruited independently to pericentromeric heterochromatin in response to hypomethylation in mouse embryonic stem cells

doi: 10.1101/2025.11.14.688451

Figure Lengend Snippet: (A) IF for Cbx7-GFP counterstained with DAPI in Dnmt1 tet/tet cells and Bend3 KO cells with or without dox. Representative nuclei from single slice of 3D stack are shown. Z-stacks obtained using 40x air objective. Scale bar corresponds to 5µm. Arrowheads point to a representative chromocenter at which the DAPI and reporter signals overlap. (B) Violin plot of mean signal intensity of Cbx7-GFP reporter signal at chromocenter normalised to that in the nucleoplasm in Dnmt1 tet/tet cells and Bend3 KO cells with or without dox. Grey dashed line corresponds the threshold above which the signal is considered increased at the chromocenter compared to the nucleoplasm. Significance was determined by Kruskal-Wallis (one-way Anova) test where **** p≤0.0001. Test was performed between every sample. Non-significant samples are not shown. (C) As in (A) IF for H2AK119ub counterstained with DAPI in Dnmt1 tet/tet cells and Bend3 KO cells with or without dox. Arrowheads point to a representative chromocenter at which the DAPI and antibody signals overlap. (D) As in (B) Violin plot of mean signal intensity of H2AK119ub antibody signal at chromocenter normalised to that in the nucleoplasm in Dnmt1 tet/tet cells and Bend3 KO cells with or without dox.

Article Snippet: Inhibition of DNMT1 protein in all other cell lines was performed using 2μM of the Dnmt1 inhibitor (MedChemExpress, #GSK-3484862) for 4 days unless otherwise stated.

Techniques: