Journal: bioRxiv

Article Title: R-loop-induced p21 expression following CDC73, CTR9, and PAF1 loss protects cancer cells against replicative catastrophe following WEE1 inhibition

doi: 10.1101/2021.07.14.452205

Figure Lengend Snippet: (A) The growth response of wild-type S. pombe , set2Δ, wee1-50, paf1Δ, set2Δ wee1-50, set2Δ paf1Δ, and set2Δ wee1-50 paf1Δ was compared by spotting 5-fold serial dilutions of each strain onto YE6S medium. Plates were the incubated for an appropriate time at either 25, 32 or 35.5 °C. ( B-C ) SETD2 WT and SETD2 CRISPR KO U2OS cells after 72 h exposure to different concentrations of AZD1775 (75, 150, 300 and 600 nM). (D-E) Viability assay to examine the impact of other PAF1 complex factors (including CDC73 and CTR9) silencing on AZD1775-sensitivity. Survival fraction of non-targeting control, CDC73, and CTR9 siRNA treated SETD2 WT and SETD2 CRISPR KO cells after 72 h exposure to different concentrations of AZD1775 (75, 150, 300 and 600 nM). Data points and bars represent the mean and SEM of ≥ three independent experiments; * P < 0.05; ** P < 0.01; and *** P < 0.001. (F) Viability assay to examine the impact of PAF1 silencing on HU or gemcitabine. Survival fraction of non-targeting control and PAF1 siRNA treated SETD2 WT and SETD2 CRISPR KO U2OS cells after 72 h exposure to different concentrations of HU (150, 300, 600 and 1200 uM) or gemcitabine (12.5, 25, 50 or 100 nM). Data points and bars represent the mean and SEM of ≥ three independent experiments; * P < 0.05; ** P < 0.01; and *** P < 0.001. (G) Viability assay to examine the sensitivity to HU in gene deletions of PAF1C components in S. pombe .

Article Snippet: Human renal cell carcinoma cell lines 786-O ( SETD2 wild-type) were a kind gift from V. Macaulay and A498 (SETD2 homozygous truncating mutation) cells were obtained from ATCC (ATCC number HTB-44).

Techniques: Incubation, CRISPR, Viability Assay, Control

Journal: bioRxiv

Article Title: R-loop-induced p21 expression following CDC73, CTR9, and PAF1 loss protects cancer cells against replicative catastrophe following WEE1 inhibition

doi: 10.1101/2021.07.14.452205

Figure Lengend Snippet: (A) Validation of PAF1 siRNA knockdown efficiency by Western Blot in SETD2 WT and SETD2 CRISPR KO U2OS cells 72 hours post-siRNA treatment. (B) Viability assay to examine the impact of PAF1 silencing on AZD1775-sensitivity. Survival fraction of non-targeting control and PAF1 siRNA treated (A-B) 786-0 ( SETD2 WT) and A498 (SETD2-mutant) renal cancer cells. C) Cleaved PARP, procaspase 3, cleaved caspase 3 and actin protein expression in SETD2 WT and SETD2 CRISPR KO U2OS cells 48 hours post-treatment with either 200 nM DMSO or 200 nM AZD1775 was assessed using Western Blot. D) Validation of CDC73 and CTR9 siRNA knockdown efficiency by Western Blot in SETD2 WT and SETD2 CRISPR KO U2OS cells 72 hours post-siRNA treatment.

Article Snippet: Human renal cell carcinoma cell lines 786-O ( SETD2 wild-type) were a kind gift from V. Macaulay and A498 (SETD2 homozygous truncating mutation) cells were obtained from ATCC (ATCC number HTB-44).

Techniques: Biomarker Discovery, Knockdown, Western Blot, CRISPR, Viability Assay, Control, Mutagenesis, Expressing

Journal: bioRxiv

Article Title: R-loop-induced p21 expression following CDC73, CTR9, and PAF1 loss protects cancer cells against replicative catastrophe following WEE1 inhibition

doi: 10.1101/2021.07.14.452205

Figure Lengend Snippet: (A) 48 hours following DMSO or AZD1775-treatment (300 nM), control or siPAF1 treated SETD2 KO U2OS cells were pulse-labelled with BrdU for 30 min and collected for cell cycle analysis. The data shown are from a single representative experiment out of three repeats. Numbers are the relative percentage of cell cycle stage. (B) Quantification of data shown in (A). (C) SETD2 CRISPR KO U2OS were treated with control or siPAF1 siRNA and exposed to 250 nM of AZD1775 for 48 hours. One representative experiment showing yH2AX vs DNA (left panel) and EdU vs DNA (right panel). The numbers indicate the percentages of cells in the high yH2AX-gated population (shown in black box). These cells correspond with the non-replicating S-phase cells as shown in the EdU plots (blue population). (D) Quantification of the yH2AX medium and high populations shown in (C). (E) Western blot analysis of SETD2 CRISPR KO U2OS cells exposed to either non-targeting control siRNA or PAF1 siRNA and/or treated with either DMSO or 300 nM AZD1775. Lamin B was used as a loading control. (F) 48 hr following AZD1775-treatment, control, siCDC73, or siCTR9 treated SETD2 KO U2OS cells were pulse-labelled with BrdU for 30 min and collected for cell cycle analysis.

Article Snippet: Human renal cell carcinoma cell lines 786-O ( SETD2 wild-type) were a kind gift from V. Macaulay and A498 (SETD2 homozygous truncating mutation) cells were obtained from ATCC (ATCC number HTB-44).

Techniques: Control, Cell Cycle Assay, CRISPR, Western Blot

Journal: bioRxiv

Article Title: R-loop-induced p21 expression following CDC73, CTR9, and PAF1 loss protects cancer cells against replicative catastrophe following WEE1 inhibition

doi: 10.1101/2021.07.14.452205

Figure Lengend Snippet: (A) SETD2 WT and (B) SETD2 CRISPR KO U2OS were treated with control, siCDC73, siCTR9 or siPAF1 and subsequently synchronized at the G 1 /S transition by a double thymidine block (DTB) and then released by addition of fresh medium. Cells were incubated with BrdU for 30 min and collected for cell cycle analysis 4, 8, and 12 hours after release. (C) Western blot analysis of RRM2 protein levels in SETD2 WT and SETD2 CRISPR KO U2OS cells exposed to either non-targeting control siRNA or PAF1 siRNA and/or treated with either DMSO or 300 nM AZD1775. Tubulin was used as a loading control.

Article Snippet: Human renal cell carcinoma cell lines 786-O ( SETD2 wild-type) were a kind gift from V. Macaulay and A498 (SETD2 homozygous truncating mutation) cells were obtained from ATCC (ATCC number HTB-44).

Techniques: CRISPR, Control, Blocking Assay, Incubation, Cell Cycle Assay, Western Blot

Journal: bioRxiv

Article Title: R-loop-induced p21 expression following CDC73, CTR9, and PAF1 loss protects cancer cells against replicative catastrophe following WEE1 inhibition

doi: 10.1101/2021.07.14.452205

Figure Lengend Snippet: (A) Western blot analysis of p21 protein levels in SETD2 WT and SETD2 CRISPR KO U2OS cells after 48 hours of treatment with either non-targeting control siRNA or PAF1 siRNA. Tubulin was used as a loading control. (B) qRT-PCR analysis of CDKN1A levels normalised to 18S in U2OS cells transfected with the indicated siRNAs (n = 3). (C) Western Blot analysis of p-CDK(S) activity following siPAF1 with and without AZD1775 in SETD2 WT U2OS cells. (D) RNA:DNA hybrid slot blot of genomic DNA ± RNaseH1 treatment from cells treated with siNT, siSETD2, siPAF1 or combined siSETD2 and siPAF1. (E) Western blot analysis of p21 protein levels in SETD2 KO U2OS cells. Immunoblot staining for the V5-epitope confirms RNAseH1 overexpression.

Article Snippet: Human renal cell carcinoma cell lines 786-O ( SETD2 wild-type) were a kind gift from V. Macaulay and A498 (SETD2 homozygous truncating mutation) cells were obtained from ATCC (ATCC number HTB-44).

Techniques: Western Blot, CRISPR, Control, Quantitative RT-PCR, Transfection, Activity Assay, Dot Blot, Staining, Over Expression

Journal: bioRxiv

Article Title: R-loop-induced p21 expression following CDC73, CTR9, and PAF1 loss protects cancer cells against replicative catastrophe following WEE1 inhibition

doi: 10.1101/2021.07.14.452205

Figure Lengend Snippet: A) 48 hr following DMSO -or AZD1775-treatment, control, siCDKN1A, siPAF1, or siCDKN1A + siPAF1 treated SETD2 KO U2OS cells were pulse-labelled with BrdU for 30 min and collected for cell cycle analysis. The data shown are from a single representative experiment out of three repeats. Numbers are the relative percentage of cell cycle stage. B) 48 hr following DMSO, AZD1775- and/or UC2288 treatment, SETD2 KO U2OS cells were collected for cell cycle analysis. C) SETD2 WT and SETD2 CRISPR KO U2OS cells after 48h exposure to different concentrations of AZD1775 (0-10 μ M) and UCC2288 (p21 inhibitor).

Article Snippet: Human renal cell carcinoma cell lines 786-O ( SETD2 wild-type) were a kind gift from V. Macaulay and A498 (SETD2 homozygous truncating mutation) cells were obtained from ATCC (ATCC number HTB-44).

Techniques: Control, Cell Cycle Assay, CRISPR

Journal: bioRxiv

Article Title: R-loop-induced p21 expression following CDC73, CTR9, and PAF1 loss protects cancer cells against replicative catastrophe following WEE1 inhibition

doi: 10.1101/2021.07.14.452205

Figure Lengend Snippet: (A) 48 hr following DMSO -or AZD1775-treatment, siPAF1, or siCDKN1A + siPAF1 treated SETD2 KO U2OS cells. The data shown are from a single representative experiment out of three repeats. Numbers are the relative percentage of cell cycle stage. (B) SETD2 CRISPR KO U2OS cells treated with siNT or siPAF1 after 48h exposure to different concentrations of AZD1775 (0-10 μM) and UCC2288 (p21 inhibitor). Survival fraction of non-targeting control, CDC73, and CTR9 (C) siRNA treated TP53 CRISPR KO cells after 72 h exposure to different concentrations of AZD1775 (75, 150, 300 and 600 nM). Data points and bars represent the mean and SEM of ≥ three independent experiments; * P < 0.05; ** P < 0.01; and *** P < 0.001. (D) Western blot analysis of p21 protein levels in SETD2 WT and SETD2 CRISPR KO U2OS cells exposed to either non-targeting control siRNA or p21 siRNA. Tubulin was used as a loading control. (E) Cell viability is further reduced when WEE1 inhibition is combined with p21 knock down in SETD2 WT and SETD2 mutant cancer cell lines. (F) Model where loss of CDC73, CTR9 and PAF1 results in resistance to WEE1i treatment.

Article Snippet: Human renal cell carcinoma cell lines 786-O ( SETD2 wild-type) were a kind gift from V. Macaulay and A498 (SETD2 homozygous truncating mutation) cells were obtained from ATCC (ATCC number HTB-44).

Techniques: CRISPR, Control, Western Blot, Inhibition, Knockdown, Mutagenesis

Journal: Epigenetics & Chromatin

Article Title: Coordinated regulation of chromatin modifiers reflects organised epigenetic programming in mouse oocytes

doi: 10.1186/s13072-025-00583-9

Figure Lengend Snippet: Primary antibodies used for indirect immunofluorescence

Article Snippet: H3K36me3 , Rabbit , 1:1000 , Validated in A498 cells with SETD2 mutation , Cell Signaling Technology, 4909S.

Techniques: Biomarker Discovery, Knock-Out, Knockdown, Mutagenesis, Membrane

Journal: Epigenetics & Chromatin

Article Title: Coordinated regulation of chromatin modifiers reflects organised epigenetic programming in mouse oocytes

doi: 10.1186/s13072-025-00583-9

Figure Lengend Snippet: SETD2 and H3K36me3 peak in early growing oocytes before declining during late oocyte growth. ( A , C ) Representative IF images showing SETD2 (A) and H3K36me3 (C) (red, grey single channel) in primordial, primary, secondary, early antral and antral follicle oocytes from wildtype adult mouse ovaries. The oocyte nucleus is defined by Lamin B1 (green, dashed lines), and DNA is shown by DAPI (blue). White boxes indicate regions shown at higher power in images on the right. Yellow arrowheads denote primordial follicle oocyte nuclei. Images are representative of multiple planes in both ovaries from three biological replicates. Scale bars represent 50 μm (left), and 10 μm (middle, right). ( B , D ) Quantification of SETD2 (B) and H3K36me3 (D) within oocyte nuclei of primordial, primary, secondary, early antral and antral follicles from wildtype adult mouse ovaries. Values represent average nuclear staining intensity with average cytoplasmic fluorescence removed to correct for non-specific background staining. Error bars represent mean ± SD. ( B ) ns: not significant, * P < 0.05, ** P < 0.01, **** P < 0.0001, one-way ANOVA plus Tukey’s multiple comparisons test. N = 94 primordial follicle oocytes, N = 56 primary follicle oocytes, N = 32 secondary follicle oocytes, N = 17 early antral follicle oocytes, and N = 13 antral follicle oocytes, in total from three biological replicates. ( D ) ns: not significant, * P < 0.05, *** P < 0.001, Kruskal-Wallis test with Dunn’s multiple comparisons test. N = 82 primordial follicle oocytes, N = 47 primary follicle oocytes, N = 21 secondary follicle oocytes, N = 11 early antral follicle oocytes, and N = 6 antral follicle oocytes, in total from three biological replicates

Article Snippet: H3K36me3 , Rabbit , 1:1000 , Validated in A498 cells with SETD2 mutation , Cell Signaling Technology, 4909S.

Techniques: Staining, Fluorescence

Journal: Epigenetics & Chromatin

Article Title: Coordinated regulation of chromatin modifiers reflects organised epigenetic programming in mouse oocytes

doi: 10.1186/s13072-025-00583-9

Figure Lengend Snippet: Loss of EED significantly increased SETD2 in primary, secondary, and early antral follicle oocytes. ( A ) Representative IF images showing SETD2 (red, grey single channel) in primary, secondary, early antral and antral follicle oocytes from Eed -wt (left), Eed -het (middle), and Eed -hom (right) adult mouse ovaries. The oocyte nucleus is defined by Lamin B1 (green, dashed lines), and DNA is shown by DAPI (blue). Images are representative of multiple planes in both ovaries from three biological replicates for each genotype. Scale bars represent 50 μm. ( B-E ) Quantification of SETD2 within oocyte nuclei of primary ( B ), secondary ( C ), early antral ( D ), and antral ( E ) follicles from Eed -wt, Eed -het, and Eed -hom adult mouse ovaries. Data is from three biological replicates for each genotype. Values represent average nuclear staining intensity with average cytoplasmic fluorescence removed to correct for non-specific background staining. Average intensity for Eed -het and Eed -hom samples are shown relative to Eed -wt set to 1.0. Error bars represent mean ± SD. ( B-C ) N = 56 Eed -wt, N = 44 Eed -het, N = 39 Eed -hom primary follicle oocytes. N = 32 Eed -wt, N = 17 Eed -het, N = 31 Eed -hom secondary follicle oocytes. ns: not significant, ** P < 0.01, Kruskal-Wallis test with Dunn’s multiple comparisons test. ( D-E ) N = 17 Eed -wt, N = 11 Eed -het, N = 6 Eed -hom early antral follicle oocytes. N = 13 Eed -wt, N = 10 Eed -het, N = 18 Eed -hom antral follicle oocytes. ns: not significant, * P < 0.05, one-way ANOVA plus Tukey’s multiple comparisons test

Article Snippet: H3K36me3 , Rabbit , 1:1000 , Validated in A498 cells with SETD2 mutation , Cell Signaling Technology, 4909S.

Techniques: Staining, Fluorescence