anti cpsf73 antibody  (Bethyl)

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: The transcription termination inhibitor JTE-607 synergistically reduces cell survival in combination with adavosertib. (A) Flow cytometry analysis of HeLa cells without (-) or with the indicated concentrations of JTE-607 and adavosertib (AZD1775) for 24 h. Numbers indicate γH2AX-positive cells. (B) Quantification of the relative proportion of γH2AX-positive cells from three independent experiments as in A. (C) Flow cytometry analysis of extracted cells as in of pRNAPII S2 levels on chromatin in HeLa cells without (-) or with 1 µM JTE-607 for 2, 6, and 24 h (orange color). Barcoded mock cells are shown in black color. (D) Quantifications from experiments, as in C. Median levels of pRNAPIIS2, RNAPII, and pRNAPII S5 were measured in G1 cells (determined based on DNA content as in ), and divided by the median levels in the barcoding control cells in each sample. Levels are shown relative to the untreated (-) G1 cells. n = 3. (E) Flow cytometry analysis as in Fig. – of non-treated cells (-) or cells treated with JTE-607 for 6 h (6h JTE-607). n = 3. (F) Images of pRNAPII S2/PCNA PLA foci in S-phase HeLa cells (PCNA-positive), with nuclei counterstained with Hoechst. Bottom panels show box plots of pRNAPII S2/PCNA and pRNAPII S5/PCNA PLA foci per S-phase cell. Cells were untreated, exposed to 1 µM JTE-607 for 4 h, to 1 µM AZD1775 for 20 min, or to both (1 µM JTE-607 for 4 h with 1 µM AZD1775 added during the last 20 min). Data are from a representative experiment. (G) Bar charts show the average of the median PLA foci obtained from three independent experiments, as in F. (H) Viability relative to DMSO control from Cell Titer Glow assay of HeLa cells 5 days after treatment with the indicated concentrations of JTE-607 and AZD1775 for 24 h before wash-off and medium replacement. (I) Synergy score (excess over BLISS) calculated from viability data from H. (J) Clonogenic survival of HeLa cells after 24-h treatment with the indicated concentrations of JTE-607 and adavosertib. Data represent the mean of three independent experiments, each performed in triplicate. (Statistical testing of synergy is shown in .) Error bars represent SEM of ≥three independent experiments;* P < 0.05; ** P < 0.01; and *** P < 0.001.

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: Flow Cytometry, Control

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

Journal: PLOS ONE

Article Title: Isoginkgetin and Madrasin are poor splicing inhibitors

doi: 10.1371/journal.pone.0310519

Figure Lengend Snippet: (A) Schematic of the mNET-seq technique. (B) Metagene profile of total pol II mNET-seq treated with DMSO (blue) or 90 μM Madrasin (red) for 30 min on scaled expressed protein-coding genes. (C) qRT-PCR with primers amplifying different protein-coding genes. HeLa cells were treated with DMSO or 90 μM Madrasin for 30 min (red) or 60 min (orange). cDNA was generated with random hexamers. Values are normalised to the 7SK snRNA and shown as relative to DMSO, mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, ** < 0.01, *** < 0.001. (D) Screenshot of the genome browser total pol II mNET-seq DMSO (blue) and Madrasin (red) tracks on the protein-coding gene KPNB1 . The arrow indicates the sense of transcription. (E) Total pol II, SPT5, CDC73, and CPSF73 ChIP-qPCR across the protein-coding gene KPNB1 in HeLa cells treated with DMSO (blue) or 90 μM Madrasin for 30 min (red). Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, ** < 0.01, *** < 0.001. (F) Ratios of SPT5 / total pol II, CDC73 / total pol II, or CPSF73 / total pol II from ChIP-qPCR on the intron-containing gene KPNB1 in HeLa cells treated with DMSO (blue) or 90 μM Madrasin for 30 min (red). Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001.

Article Snippet: For each IP, 60 μg of chromatin was incubated overnight on a rotating wheel at 4°C with the following antibodies: normal rabbit IgG (2729S, Cell Signaling), RNA polymerase II antibody (NBP2-32080, Novus Biologicals), Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (13499S, Cell Signaling Technology), Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb (13523S, Cell Signaling Technology), CDC73 (A300-170A, Bethyl Laboratories), SUPT5H antibody (A300-868A, Bethyl Laboratories), and CPSF73 (A301-091A, Bethyl Laboratories).

Techniques: Quantitative RT-PCR, Generated, Two Tailed Test, ChIP-qPCR

Journal: PLOS ONE

Article Title: Isoginkgetin and Madrasin are poor splicing inhibitors

doi: 10.1371/journal.pone.0310519

Figure Lengend Snippet: (A) Metagene profiles of total pol II mNET-seq performed in HeLa cells treated with DMSO (blue) or 90 μM Madrasin for 30 min (red) on scaled expressed intronless genes. (B) Screenshot of the genome browser total pol II mNET-seq DMSO (blue) and Madrasin (red) tracks of the intronless gene JUN . The arrow indicates the sense of transcription. (C) Total pol II, SPT5, CDC73, and CPSF73 ChIP-qPCR on the intronless gene JUN in HeLa cells treated with DMSO (blue) or 90 μM Madrasin for 30 min (red). Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001. (D) Metagene profiles of total pol II mNET-seq performed in HeLa cells treated with DMSO (blue) or 90 μM Madrasin for 30 min (red) on scaled expressed histone genes. (E) Screenshot of the genome browser total pol II mNET-seq DMSO (blue) and Madrasin (red) tracks of the histone gene H1-2 . The arrow indicates the sense of transcription. (F) Total pol II, SPT5, CDC73, and CPSF73 ChIP-qPCR on the histone gene H1-2 in HeLa cells treated with DMSO (blue) or 90 μM Madrasin for 30 min (red). Mean ± SEM, n = 3 biological replicates. Statistical test: two-tailed unpaired t-test. P-value: * < 0.05.

Article Snippet: For each IP, 60 μg of chromatin was incubated overnight on a rotating wheel at 4°C with the following antibodies: normal rabbit IgG (2729S, Cell Signaling), RNA polymerase II antibody (NBP2-32080, Novus Biologicals), Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb (13499S, Cell Signaling Technology), Phospho-Rpb1 CTD (Ser5) (D9N5I) Rabbit mAb (13523S, Cell Signaling Technology), CDC73 (A300-170A, Bethyl Laboratories), SUPT5H antibody (A300-868A, Bethyl Laboratories), and CPSF73 (A301-091A, Bethyl Laboratories).

Techniques: ChIP-qPCR, Two Tailed Test

Journal: Cell reports

Article Title: Non-canonical isoforms of the mRNA polyadenylation factor WDR33 regulate STING-mediated immune responses

doi: 10.1016/j.celrep.2024.113886

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-CPSF73 , Abclonal , Cat# A2222.

Techniques: Virus, Recombinant, Negative Control, Software