Journal: Nucleic Acids Research

Article Title: Transcription termination counteracts DNA damage after WEE1 inhibition

doi: 10.1093/nar/gkaf1487

Figure Lengend Snippet: WDR82/PNUTS-PP1 is required to suppress DNA damage in S-phase after WEE1 inhibition. (A) Flow cytometry analysis of control siRNA (scr) or WDR82 siRNA (siWDR82) transfected HeLa cells treated with or without 1 µM AZD1775 for 2 or 6 h and stained with the DNA damage marker γH2AX and the DNA stain Hoechst 33258. Dotted lines indicate S-phase. The bar chart shows quantification of median γH2AX levels in S-phase from three independent experiments. (B) Western blot analysis of HeLa cells (HeLa) and HeLa cells stably expressing siRNA-resistant WDR82 (WDR82-res). Cells were siRNA-transfected and treated as in A. (C) Quantification of pATM T1989, pDNA-PK S2056, and pRPA S4/S8 relative to levels of CDK1 from six (HeLa) or three (WDR82-res) independent experiments, as in B. (D) Flow cytometry analysis of scr or PNUTS siRNA (siPNUTS) transfected cells treated with or without 1 µM AZD1775 for 2 h at 48 h after siRNA transfection, and stained with anti-γH2AX and Hoechst 33258. The bar chart shows quantification of median γH2AX levels in S-phase from three independent experiments. (E) Relative viability of scr and siWDR82 transfected cells treated with indicated concentrations of adavosertib (AZD1775) for 24 h, assessed by the Cell Titer Glow assay four days later. Inhibitors were added 48 h post-transfection ( n = 3). Error bars: SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (two tailed Student’s t-test).

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775, Selleck Biochem) and the CPSF73 inhibitor JTE-607 (MedChemExpress) were used at indicated concentrations, the CDK9 inhibitor 5,6-dichloro-1-beta-ribo-furanosyl benzimidazole (DRB) (Sigma Aldrich) was used at 100 μM, the CDK7-inhibitor THZ1 (ApexBio) at 1 μM, the XPB inhibitor Triptolide (TPL) (Sigma Aldrich) at 1 μM, and the CHK1 inhibitor AZD7762 (Selleckchem) at 300 nM.

Techniques: Inhibition, Flow Cytometry, Control, Transfection, Staining, Marker, Western Blot, Stable Transfection, Expressing, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Transcription termination counteracts DNA damage after WEE1 inhibition

doi: 10.1093/nar/gkaf1487

Figure Lengend Snippet: Inhibitors of active transcription or depletion of transcription elongation factor CDC73 suppress DNA damage after WEE1 inhibition. (A) Western blot analysis of scr and siWDR82 transfected cells with and without 1 µM adavosertib (AZD1775) for 2 or 6 h. Transcription inhibitors THZ1 and DRB were added 30 min prior to AZD1775. (B) Quantifications from experiments as in A, showing pDNA-PK S2056 and pRPA S4S8 versus CDK1. n = 3 for DRB-treated and n = 2 for THZ1-treated samples. (C) Flow cytometry analysis of cells treated with 1 µM AZD1775 at 72h after siRNA transfection and harvested 6 h later. The bar chart shows quantification of S-phase levels of γH2AX from three independent experiments. (D) Readthrough analysis from chromatin RNA sequencing experiment of HeLa cells 48 h after siRNA transfection. Box plot showing Log2 ratio of readthrough versus gene from the top 2000 expressed genes. (E) Read density profiles from experiments performed as in D showing readthrough from the gene with the biggest difference in readthrough between siWDR82 and scr, visualized in IGV ( VDAC2 : chr10:75 229 310). (F) Left: Readthrough analysis from chromatin RNA sequencing as in D for all expressed genes (defined as described in materials and methods). Middle: Readthrough analysis for expressed genes with detectable readthrough (log2 ratio > –5). Right: number of expressed genes with log2 ratio >–2. (G) Quantifications of pRNAPII S2 levels on chromatin from flow cytometry analysis, as in . Median levels in segments (shown in ) were divided by pRNAPII S2 levels in the internal barcoding control cells. Thereafter, median levels in each segment of the respective siRNA condition (scr or siCDC73) were divided by the median levels in segment 1 (G1). n = 6. (H) As in G, but for scr, siWDR82, and siWDR82 + siCDC73. n = 3. (I) As in G, but for scr, siPNUTS, and siPNUTS + siCDC73. n = 3. Error bars: SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (two tailed Student’s t-test). Statistical comparisons in G-I: scr versus siCDC73/siWDR82/siPNUTS; siWDR82 + siCDC73 versus siWDR82; siPNUTS + siCDC73 versus siPNUTS. Results in D-F are from a representative experiment.

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775, Selleck Biochem) and the CPSF73 inhibitor JTE-607 (MedChemExpress) were used at indicated concentrations, the CDK9 inhibitor 5,6-dichloro-1-beta-ribo-furanosyl benzimidazole (DRB) (Sigma Aldrich) was used at 100 μM, the CDK7-inhibitor THZ1 (ApexBio) at 1 μM, the XPB inhibitor Triptolide (TPL) (Sigma Aldrich) at 1 μM, and the CHK1 inhibitor AZD7762 (Selleckchem) at 300 nM.

Techniques: Inhibition, Western Blot, Transfection, Flow Cytometry, RNA Sequencing, Control, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Transcription termination counteracts DNA damage after WEE1 inhibition

doi: 10.1093/nar/gkaf1487

Figure Lengend Snippet: Transcription termination suppresses DNA damage in S-phase after WEE1 inhibition. (A) Flow cytometry analysis of cells transfected with scr and siRNA against DDX5 (siDDX5) and XRN2 (siXRN2) with and without 1 µM adavosertib (AZD1775) for 2 h, at 48 h after siRNA transfection. (B) As in A, but cells were transfected with siRNA against CPSF73 (siCPSF73). (C) As in A, but cells were transfected with scr, siWDR82, and siCPSF73 with and without siRNA against CDC73 (siCDC73). Of note is that cells transfected with siCDC73 in the absence of siWDR82 and siCPSF73 were transfected with only siCDC73 (not in combination with scr). (D) Bar chart showing quantifications of median γH2AX levels in S-phase from experiments as in A–C. Error bars represent SEM from ≥3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 (two-tailed Student’s t-test, except one-sample t-test for comparisons to the normalized sample). (E) Western blot showing knockdown levels 48 h after siRNA transfection using the different siRNAs in A–D. (F) As in E. Error bars: SEM of ≥3 independent experiments;* P < 0.05; ** P < 0.01; and *** P < 0.001 (two-tailed Student’s t-test).

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775, Selleck Biochem) and the CPSF73 inhibitor JTE-607 (MedChemExpress) were used at indicated concentrations, the CDK9 inhibitor 5,6-dichloro-1-beta-ribo-furanosyl benzimidazole (DRB) (Sigma Aldrich) was used at 100 μM, the CDK7-inhibitor THZ1 (ApexBio) at 1 μM, the XPB inhibitor Triptolide (TPL) (Sigma Aldrich) at 1 μM, and the CHK1 inhibitor AZD7762 (Selleckchem) at 300 nM.

Techniques: Inhibition, Flow Cytometry, Transfection, Two Tailed Test, Western Blot, Knockdown

Journal: Nucleic Acids Research

Article Title: Transcription termination counteracts DNA damage after WEE1 inhibition

doi: 10.1093/nar/gkaf1487

Figure Lengend Snippet: Combined targeting of transcription termination with WEE1 synergistically reduces cell survival in prostate cancer cells. (A) Kaplan-Meier plot showing BCR-free survival of prostate cancer patients vs time after prostatectomy. Patients were grouped into two by the median CPSF73 expression based on Illumina Bead Array gene expression data. n = 93. (B) CPSF73 levels versus ISUP grade group (left) and pathological tumor stage (right) in prostate cancer patients. n = 94. (C) Flow cytometry analysis of DU145 cells (left) and PC3 cells (right) without (-) or with JTE-607 (1 µM) and adavosertib (AZD1775) (500 nM) for 24 h. Regions indicate percentage cells with high γH2AX levels. (D) Quantifications from experiments, as in C, showing average % cells with high γH2AX as determined by the regions shown in C. Results are shown relative to the percentage of cells with high γH2AX after treatment with JTE-607 and AZD1775. n = 3. (E) Average viability relative to DMSO control from Cell Titer Glow assays of DU145 cells (left) and PC3 cells (right) treated with the indicated concentrations of JTE-607 and AZD1775 for 5 days. n = 3. (F) Average synergy score (excess over BLISS) calculated from viability data from E. (G) Clonogenic survival of DU145 cells (left) and PC3 cells (right) after treatment with the indicated concentrations of JTE-607 and adavosertib. Data represent the mean of three independent experiments, each performed in triplicate. (H) Working model. See main text for details. Error bars represent SEM of ≥three independent experiments. * P < 0.05; ** P < 0.01; and *** P < 0.001. Statistical testing of synergy in F and G is shown in and .

Article Snippet: The WEE1 inhibitor adavosertib (AZD1775, Selleck Biochem) and the CPSF73 inhibitor JTE-607 (MedChemExpress) were used at indicated concentrations, the CDK9 inhibitor 5,6-dichloro-1-beta-ribo-furanosyl benzimidazole (DRB) (Sigma Aldrich) was used at 100 μM, the CDK7-inhibitor THZ1 (ApexBio) at 1 μM, the XPB inhibitor Triptolide (TPL) (Sigma Aldrich) at 1 μM, and the CHK1 inhibitor AZD7762 (Selleckchem) at 300 nM.

Techniques: Expressing, Gene Expression, Flow Cytometry, Control