Journal: Reproductive sciences (Thousand Oaks, Calif.)

Article Title: A Preliminary Study: Human Fibroid Stro-1 + /CD44 + Stem Cells Isolated From Uterine Fibroids Demonstrate Decreased DNA Repair and Genomic Integrity Compared to Adjacent Myometrial Stro-1 + /CD44 + Cells.

doi: 10.1177/1933719118783252

Figure Lengend Snippet: Figure 8. Altered initiation of DNA damage response (DDR) in fibroid (F) stem cells: decreased DDR signal amplification and transduction following DNA double-strand break (DSB) induction. A, Western blots and corresponding densitometry of sensors of DNA DSBs, p-MRE11 (Ser676), MRE11, p-NBS1 (Ser343), and NBS1: untreated (UT), bleomycin-treated þ 1, 6, and 24 hours of recovery in n ¼ 4 patients. B, Western blots and corresponding densitometry of signal-transducing kinases, phosphorylated-Ataxia Telangiectasia Mutated (p-ATM; Ser1981), ATM, p-ATR (Ser1989), ATR: UT, bleomycin-treated þ 1, 6, and 24 hours of recovery in n ¼ 4 patients. C, Western blots and corresponding densitometry of effector kinases, p-CHK1 (Ser345), CHK1, p-CHK2 (Thr68), CHK2: UT, bleomycin-treated þ 1, 6, and 24 hours of recovery in n ¼ 4 patients. For each time point, the mean expression ratio in F: Myo was compared to 1 using a 2-sided 1-sample t test. Lines represent means + standard error of the meam. *P < .05. p-CHK1 indicates phosphorylated-Checkpoint Kinase 1; p-MRE11, phosphorylated-MRE11; p- NBS1, phosphorylated-Nijmegen Breakage Syndrome 1; p-ATR, phosphorylated-Ataxia Telangiectasia.

Article Snippet: Membranes were then incubated with primary antibodies (Supplemental Table S2): rabbit anti-phosphoATM (Ser1981), rabbit anti-ATM, rabbit anti-phospho-ATR (Thr1989), rabbit anti-ATR, rabbit anti-phospho-CHK1 (Ser345), mouse anti-CHK1, rabbit anti-phospho-CHK2 (Thr68), mouse anti-CHK2, rabbit anti-phospho-MRE11 (Ser676), rabbit anti-MRE11, rabbit anti-phospho-p95/NBS1 (Ser343), rabbit anti-p95/NBS1, and rabbit anti-RAD50 (all antibodies: Cell Signaling, Danvers, Massachusetts; 1:1000 dilution) overnight at 4 C in either 1% wt/vol nonfat dry milk or 1% BSA in 0.1% PBS-Tween (PBS-T) per antibody specification.

Techniques: Amplification, Transduction, Western Blot, Expressing

Journal: Carcinogenesis

Article Title: Triggering of transient receptor potential vanilloid type 1 (TRPV1) by capsaicin induces Fas/CD95-mediated apoptosis of urothelial cancer cells in an ATM-dependent manner.

doi: 10.1093/carcin/bgp138

Figure Lengend Snippet: Fig. 5. CPS induces ATM activation that is involved in the control of p53 phosphorylation, Fas/CD95 expression and clustering and RT4 cell growth and apoptosis. (A) Lysates from RT4 cells treated at different times with 100 lM CPS were separated on 6 and 12% SDS–PAGE and probed with rabbit anti-phospho ATM (Ser1981) Ab or with a rabbit anti-human p53, phospho-p53 Ser15, phospho-p53 Ser20 or phospho-p53 Ser392 Abs, respectively. a-Tubulin levels were evaluated as protein loading control. Data are representative of three separate experiments. (B) Lysates from RT4 cells treated for 24 h with 100 lM CPS alone or in combination with 10 lM KU55933 were probed with rabbit anti-human phospho-p53 Ser15, Ser20 and Ser392 Abs. a-Tubulin levels were analyzed as protein loading control. Data are representative of three separate experiments. (C) Fas/CD95 expression was evaluated in RT4 cells treated for 12 and 24 h with 100 lM CPS alone or in combination with 10 lM KU55933, by immunofluorescence and FACS analysis using an anti-Fas/CD95 mAb followed by a phycoerythrin- conjugated GAM. Data are representative of three different experiments. (D) The immunocytochemical localization of Fas/CD95 and TRPV1 in RT4 cells treated for 24 h with 100 lM of CPS alone or in combination with 10 lM of KU55933 was evaluated as described in Figure 5 panel C; bar 5 10 lm. (E) Cell growth was evaluated by MTT assay in RT4 cells treated for 24 h with 100 lM CPS and 10 lM KU55933 alone or in combination. Control sample indicates CPS and/or KU55933 vehicles treatment. Statistical analysis was performed comparing KU55933- or CPS-treated RT4 cells with control and CPS plus KU55933- with CPS- treated cells; P , 0.01. (F) Annexin-positive RT4 cells treated for 24 h with 100 lM CPS alone or in combination with 10 lM KU55933 was evaluated as described in Figure 3 panel D. Data are representative of three separate experiments.

Article Snippet: The following antibodies (Abs) were used: goat anti-human TRPV1 from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit anti-human p53, rabbit anti-human phospho-p53 (Ser20), rabbit anti-human phospho-p53 (Ser392) and rabbit anti-human BID from Cell Signaling (Danvers, MA); rabbit antihuman caspase-3 and caspase-9 from Calbiochem–Novabiochem Corporation (San Diego, CA) and rabbit anti-human phosphoATM (Ser1981) from RD Systems (South Beloit, IL).

Techniques: Activation Assay, Control, Phospho-proteomics, Expressing, SDS Page, MTT Assay

Journal: bioRxiv

Article Title: Regulation of ATR activity by the RNA polymerase II phosphatase PNUTS-PP1

doi: 10.1101/267013

Figure Lengend Snippet: A) Western blot analysis of ATR and ATM signaling events in control scrambled siRNA transfected (scr) or PNUTS siRNA transfected (siPNUTS #1 and siPNUTS) HeLa cells, at indicated times after 10 Gy. Cells were harvested at 72 hours after siRNA transfection. Bottom bar charts show quantification of pCHK1 S317 relative to CHK1 and pRPA S33 relative to γTUBULIN levels (n=8). B) Western blot analysis at 2 or 6 hr after addition of thymidine to cells siRNA transfected as in A) (scr and siPNUTS). Bottom bar charts show quantification of pCHK1 S317 relative to CHK1 and pRPA S33 relative to RPA70 levels (n=10). C) Western blot analysis of HeLa cells or HeLa BAC clones stably expressing EGFP mouse pnuts (mpnuts) transfected with scr or siPNUTS (specifically targets human PNUTS), at 1 or 6 hr after 10 Gy. Lines to the right of the western blot indicate migration of human endogenous PNUTS (lower band) and EGFP mpnuts (upper band). Bottom bar chart shows quantification of pCHK1 S345 relative to CHK1 levels (n=3). D) Western blot analysis of HeLa cells transfected with scr or siPNUTS. At 24 hr post transfection, the indicated samples were transfected with wild type EGFP PNUTS or PP1-binding deficient EGFP PNUTS RAXA. Cells were harvested 48 hr later without further treatment (-) or 1 hr after 10 Gy. Lines to the right of the western blot indicate migration of endogenous PNUTS (lower band) and EGFP PNUTS/EGFP PNUTS RAXA (upper band), asterisk indicates what is likely EGFP PNUTS/EGFP PNUTS RAXA degradation products. Bar chart shows quantification of pCHK1 S317 relative to CHK1 (n=3).

Article Snippet: Antibodies were: anti-PNUTS (BD Biosciences), anti-phosphoCHK1 Ser317, anti-phosphoCHK1 Ser345, anti-phosphoATM Ser1981, anti-phosphoCHK2 Thr68, anti-ATR (all from Cell Signaling Technology), anti-CHK1 and anti-CHK2 (both ( )), anti-γTUBULIN (GTU-88, Sigma), anti-CDK1 (sc-54), anti-RNAPII (F-12), anti-MCM7 (DCS-141) (all from Santa Cruz Biotechnology), anti-phospho RPA32 Ser33, anti-phosphoDNAPK S2056 (both from Abcam), anti-phospho RNAPII S5 (3E8), anti-phospho RNAPII S7 (4E12), anti-phospho RNAPII S2 (3E10) (all from Millipore), anti-CDC73 (Bethyl), anti-phosphoATR Thr1989 (GeneTex) and anti-ETAA1 ( ).

Techniques: Western Blot, Transfection, Clone Assay, Stable Transfection, Expressing, Migration, Binding Assay

Journal: bioRxiv

Article Title: Regulation of ATR activity by the RNA polymerase II phosphatase PNUTS-PP1

doi: 10.1101/267013

Figure Lengend Snippet: A) Western blot analysis and quantifications of scr, siPNUTS or CDC73 siRNA (siCDC73 #2) transfected HeLa cells or HeLa cells stably expressing siRNA-resistant Flag-CDC73 treated with IR (10Gy) as indicated. Bottom bar charts show quantification of pCHK1 S345 and pCHK1317 versus CHK1 levels at 6 hours after 10 Gy (n=3). *indicates p<0.05 by two-tailed T-test. B) Western blot analysis of immunoprecipitations from HeLa cell lysates, using a control antibody (ctrl Ab), no antibody (beads) or anti-CDC73 antibodies (aCDC73 IP). *Indicates IgG band from the control antibody, which migrated slightly faster than CDC73 in the western blot. C) Western blot analysis of immunoprecipitations as in B, but using anti-pATR T1989 antibodies(apATR T1989 IP). D) Model for how PNUTS-PP1 may suppress ATR signaling. Based on our results, we propose a pathway where RNAPII-CTD signals to ATR via CDC73. PNUTS-PP1 counteracts this by dephosphorylating the CTD. Furthermore, our results indicate that TOPBP1 and ETAA1 can direct the ATR activity towards pCHK1 S317/345 and pRPA S33, respectively.

Article Snippet: Antibodies were: anti-PNUTS (BD Biosciences), anti-phosphoCHK1 Ser317, anti-phosphoCHK1 Ser345, anti-phosphoATM Ser1981, anti-phosphoCHK2 Thr68, anti-ATR (all from Cell Signaling Technology), anti-CHK1 and anti-CHK2 (both ( )), anti-γTUBULIN (GTU-88, Sigma), anti-CDK1 (sc-54), anti-RNAPII (F-12), anti-MCM7 (DCS-141) (all from Santa Cruz Biotechnology), anti-phospho RPA32 Ser33, anti-phosphoDNAPK S2056 (both from Abcam), anti-phospho RNAPII S5 (3E8), anti-phospho RNAPII S7 (4E12), anti-phospho RNAPII S2 (3E10) (all from Millipore), anti-CDC73 (Bethyl), anti-phosphoATR Thr1989 (GeneTex) and anti-ETAA1 ( ).

Techniques: Western Blot, Transfection, Stable Transfection, Expressing, Two Tailed Test, Activity Assay

Journal: bioRxiv

Article Title: Regulation of ATR activity by the RNA polymerase II phosphatase PNUTS-PP1

doi: 10.1101/267013

Figure Lengend Snippet: A) Western blot analysis of ATR and ATM signaling events in control scrambled siRNA transfected (scr) or PNUTS siRNA transfected (siPNUTS #1 and siPNUTS) HeLa cells, at indicated times after 10 Gy. Cells were harvested at 72 hours after siRNA transfection. Bottom bar charts show quantification of pCHK1 S317 relative to CHK1 and pRPA S33 relative to γTUBULIN levels (n=8). B) Western blot analysis at 2 or 6 hr after addition of thymidine to cells siRNA transfected as in A) (scr and siPNUTS). Bottom bar charts show quantification of pCHK1 S317 relative to CHK1 and pRPA S33 relative to RPA70 levels (n=10). C) Western blot analysis of HeLa cells or HeLa BAC clones stably expressing EGFP mouse pnuts (mpnuts) transfected with scr or siPNUTS (specifically targets human PNUTS), at 1 or 6 hr after 10 Gy. Lines to the right of the western blot indicate migration of human endogenous PNUTS (lower band) and EGFP mpnuts (upper band). Bottom bar chart shows quantification of pCHK1 S345 relative to CHK1 levels (n=3). D) Western blot analysis of HeLa cells transfected with scr or siPNUTS. At 24 hr post transfection, the indicated samples were transfected with wild type EGFP PNUTS or PP1-binding deficient EGFP PNUTS RAXA. Cells were harvested 48 hr later without further treatment (-) or 1 hr after 10 Gy. Lines to the right of the western blot indicate migration of endogenous PNUTS (lower band) and EGFP PNUTS/EGFP PNUTS RAXA (upper band), asterisk indicates what is likely EGFP PNUTS/EGFP PNUTS RAXA degradation products. Bar chart shows quantification of pCHK1 S317 relative to CHK1 (n=3).

Article Snippet: Antibodies were: anti-PNUTS (BD Biosciences), anti-phosphoCHK1 Ser317, anti-phosphoCHK1 Ser345, anti-phosphoATM Ser1981, anti-phosphoCHK2 Thr68, anti-ATR (all from Cell Signaling Technology), anti-CHK1 and anti-CHK2 (both ( )), anti-γTUBULIN (GTU-88, Sigma), anti-CDK1 (sc-54), anti-RNAPII (F-12), anti-MCM7 (DCS-141) (all from Santa Cruz Biotechnology), anti-phospho RPA32 Ser33, anti-phosphoDNAPK S2056 (both from Abcam), anti-phospho RNAPII S5 (3E8), anti-phospho RNAPII S7 (4E12), anti-phospho RNAPII S2 (3E10) (all from Millipore), anti-CDC73 (Bethyl), anti-phosphoATR Thr1989 (GeneTex) and anti-ETAA1 ( ).

Techniques: Western Blot, Transfection, Clone Assay, Stable Transfection, Expressing, Migration, Binding Assay

Journal: bioRxiv

Article Title: Regulation of ATR activity by the RNA polymerase II phosphatase PNUTS-PP1

doi: 10.1101/267013

Figure Lengend Snippet: A) Western blot analysis and quantifications of scr, siPNUTS or CDC73 siRNA (siCDC73 #2) transfected HeLa cells or HeLa cells stably expressing siRNA-resistant Flag-CDC73 treated with IR (10Gy) as indicated. Bottom bar charts show quantification of pCHK1 S345 and pCHK1317 versus CHK1 levels at 6 hours after 10 Gy (n=3). *indicates p<0.05 by two-tailed T-test. B) Western blot analysis of immunoprecipitations from HeLa cell lysates, using a control antibody (ctrl Ab), no antibody (beads) or anti-CDC73 antibodies (aCDC73 IP). *Indicates IgG band from the control antibody, which migrated slightly faster than CDC73 in the western blot. C) Western blot analysis of immunoprecipitations as in B, but using anti-pATR T1989 antibodies(apATR T1989 IP). D) Model for how PNUTS-PP1 may suppress ATR signaling. Based on our results, we propose a pathway where RNAPII-CTD signals to ATR via CDC73. PNUTS-PP1 counteracts this by dephosphorylating the CTD. Furthermore, our results indicate that TOPBP1 and ETAA1 can direct the ATR activity towards pCHK1 S317/345 and pRPA S33, respectively.

Article Snippet: Antibodies were: anti-PNUTS (BD Biosciences), anti-phosphoCHK1 Ser317, anti-phosphoCHK1 Ser345, anti-phosphoATM Ser1981, anti-phosphoCHK2 Thr68, anti-ATR (all from Cell Signaling Technology), anti-CHK1 and anti-CHK2 (both ( )), anti-γTUBULIN (GTU-88, Sigma), anti-CDK1 (sc-54), anti-RNAPII (F-12), anti-MCM7 (DCS-141) (all from Santa Cruz Biotechnology), anti-phospho RPA32 Ser33, anti-phosphoDNAPK S2056 (both from Abcam), anti-phospho RNAPII S5 (3E8), anti-phospho RNAPII S7 (4E12), anti-phospho RNAPII S2 (3E10) (all from Millipore), anti-CDC73 (Bethyl), anti-phosphoATR Thr1989 (GeneTex) and anti-ETAA1 ( ).

Techniques: Western Blot, Transfection, Stable Transfection, Expressing, Two Tailed Test, Activity Assay