Journal: Nature communications

Article Title: ARF triggers senescence in Brca2-deficient cells by altering the spectrum of p53 transcriptional targets.

doi: 10.1038/ncomms3697

Figure Lengend Snippet: Figure 2 | ARF activation in BRCA2-deficient mouse and human cells is ATM-dependent. (a) MEFs established from Brca2F/ p53 þ / þ embryos were infected three times at 12-h intervals with retroviruses expressing self-deleting Cre recombinase ( þ Cre) or control pBabe empty vector ( Cre), in the presence or absence of ATM inhibitor Ku55933. SMC1 and tubulin were used as loading controls. (b) Human H1299 cells were infected with lentiviruses expressing control or BRCA2 shRNAs, followed by selection with puromycin for 48 h. Five days after infection, cells were transfected with control or ATM siRNAs. Cell extracts were prepared 6 days after infection and were analysed using western blotting. SMC1 and GAPDH were used as loading controls. (c) ATM-deficient GM16666 human cells and ATM-expressing GM16667 cells were transfected with control or BRCA2 siRNAs twice at 3 days interval. Extracts were prepared 6 days after the first transfection and immunoblotted as indicated. GM16667 cells used as control were irradiated (10 Gy) and extracts were prepared 2 h later. SMC1 was used as a loading control. *, nonspecific band.

Article Snippet: The following antibodies were used in immunoblotting: rabbit polyclonal antisera raised against ATM (Sigma Aldrich), H2AX (Calbiochem), SMC1 (Bethyl Laboratories), CHK1 (Cell Signalling Technology), phosphorylated CHK1 (Ser 345, Cell Signalling Technology), phosphorylated CHK2 (Thr 68, Cell Signalling Technology), RAD51 (Santa Cruz Biotechnology), p14 (Abcam), p16 (Santa Cruz Biotechnology), p53 (Santa Cruz Biotechnology), ERK (Cell Signalling Technology), full-length PARP (9542, Cell Signalling Technology), cleaved PARP Asp214 (9541, Cell Signalling Technology), phospho-p53 Ser15 (Cell Signalling Technology), E2F1 (Santa Cruz Biotechnology), DUSP4 (Santa Cruz Biotechnology), DUSP7 (Abcam) and human histone H3 (a gift from A. Verreault); mouse monoclonal antibodies raised against BRCA2 (OP95, Calbiochem), CHK2/Cds1 (Upstate), P-ERK (Cell Signalling Technology), phosphorylated ATM (Cell Signalling Technology), GAPDH (Novus Biologicals), PCNA (Santa Cruz Biotechnology) and a-tubulin68 (Cancer Research UK Monoclonal Antibody Service); rat monoclonal antibody against p19 (Novus); goat polyclonal antibody raised against ATR (Santa Cruz Biotechnology) and sheep polyclonal antibody raised against BRCA2 (ref. 69).

Techniques: Activation Assay, Infection, Expressing, Control, Plasmid Preparation, Selection, Transfection, Western Blot, Irradiation

Journal: Nature communications

Article Title: ARF triggers senescence in Brca2-deficient cells by altering the spectrum of p53 transcriptional targets.

doi: 10.1038/ncomms3697

Figure Lengend Snippet: Figure 4 | ATR induces ARF expression in response to HR deficiency or oncogenic stress. (a) Human H1299 cells were infected with lentiviruses expressing indicated shRNAs, followed by selection with puromycin for 48 h. Cell extracts were prepared 6 days after infection and were analysed using western blotting. SMC1 was used as a loading control. (b) MEFs established from p53 / embryos were infected three times with control or RAD51 shRNAs, in the presence or absence of ATM or ATR chemical inhibitors (ATMi, ATRi). Extracts prepared from cells collected 6 days after the first infection were immunoblotted as indicated. Tubulin was used as a loading control. (c) MEFs established from p53 / embryos were infected with control or K-RAS-expressing retroviruses, in the presence of control or ATR shRNAs. Extracts prepared from cells collected 6 days after the first infection were immunoblotted as indicated. ERK, SMC1 and H3 were used as loading controls. (d) Quantification of replication fork speed and tract length using single DNA fibre analysis in cells treated as in c. Error bars represent s.d. of two independent experiments. The statistical significance of the observed reduction in replication tract length induced by K-RAS expression was evaluated using an unpaired two-tailed t-test. Representative images of ongoing replication forks identified with CldU and IdU staining are also shown. (e) Schematic diagram of the DNA damage response to replicative stress, leading to ARF accumulation.

Article Snippet: The following antibodies were used in immunoblotting: rabbit polyclonal antisera raised against ATM (Sigma Aldrich), H2AX (Calbiochem), SMC1 (Bethyl Laboratories), CHK1 (Cell Signalling Technology), phosphorylated CHK1 (Ser 345, Cell Signalling Technology), phosphorylated CHK2 (Thr 68, Cell Signalling Technology), RAD51 (Santa Cruz Biotechnology), p14 (Abcam), p16 (Santa Cruz Biotechnology), p53 (Santa Cruz Biotechnology), ERK (Cell Signalling Technology), full-length PARP (9542, Cell Signalling Technology), cleaved PARP Asp214 (9541, Cell Signalling Technology), phospho-p53 Ser15 (Cell Signalling Technology), E2F1 (Santa Cruz Biotechnology), DUSP4 (Santa Cruz Biotechnology), DUSP7 (Abcam) and human histone H3 (a gift from A. Verreault); mouse monoclonal antibodies raised against BRCA2 (OP95, Calbiochem), CHK2/Cds1 (Upstate), P-ERK (Cell Signalling Technology), phosphorylated ATM (Cell Signalling Technology), GAPDH (Novus Biologicals), PCNA (Santa Cruz Biotechnology) and a-tubulin68 (Cancer Research UK Monoclonal Antibody Service); rat monoclonal antibody against p19 (Novus); goat polyclonal antibody raised against ATR (Santa Cruz Biotechnology) and sheep polyclonal antibody raised against BRCA2 (ref. 69).

Techniques: Expressing, Infection, Selection, Western Blot, Control, Two Tailed Test, Staining

Journal: Nature communications

Article Title: ARF triggers senescence in Brca2-deficient cells by altering the spectrum of p53 transcriptional targets.

doi: 10.1038/ncomms3697

Figure Lengend Snippet: Figure 3 | MRE11-dependent ssDNA formation triggers ARF induction in BRCA2- and RAD51-deficient cells. (a) Human H1299 cells were infected with lentiviruses expressing control or BRCA2 shRNAs, followed by selection with puromycin for 48 h, in the presence or absence of MRE11 inhibitor mirin. Single-stranded DNA in these cells was quantified using FACS analysis of BrdU immunofluorescence detected under non-denaturing conditions. (b) Cell extracts from cells treated as in a were prepared 6 days after infection and analysed using western blotting. SMC1 and H2AX were used as loading controls. (c) MEFs established from p53 / embryos were treated with GFP control or RAD51 shRNAs followed by selection with puromycin for 48 h, in the presence or absence of MRE11 inhibitor mirin. Extracts were prepared from cells collected 6 days after infection and immunoblotted as indicated. Histone H2AX was used as a loading control.

Article Snippet: The following antibodies were used in immunoblotting: rabbit polyclonal antisera raised against ATM (Sigma Aldrich), H2AX (Calbiochem), SMC1 (Bethyl Laboratories), CHK1 (Cell Signalling Technology), phosphorylated CHK1 (Ser 345, Cell Signalling Technology), phosphorylated CHK2 (Thr 68, Cell Signalling Technology), RAD51 (Santa Cruz Biotechnology), p14 (Abcam), p16 (Santa Cruz Biotechnology), p53 (Santa Cruz Biotechnology), ERK (Cell Signalling Technology), full-length PARP (9542, Cell Signalling Technology), cleaved PARP Asp214 (9541, Cell Signalling Technology), phospho-p53 Ser15 (Cell Signalling Technology), E2F1 (Santa Cruz Biotechnology), DUSP4 (Santa Cruz Biotechnology), DUSP7 (Abcam) and human histone H3 (a gift from A. Verreault); mouse monoclonal antibodies raised against BRCA2 (OP95, Calbiochem), CHK2/Cds1 (Upstate), P-ERK (Cell Signalling Technology), phosphorylated ATM (Cell Signalling Technology), GAPDH (Novus Biologicals), PCNA (Santa Cruz Biotechnology) and a-tubulin68 (Cancer Research UK Monoclonal Antibody Service); rat monoclonal antibody against p19 (Novus); goat polyclonal antibody raised against ATR (Santa Cruz Biotechnology) and sheep polyclonal antibody raised against BRCA2 (ref. 69).

Techniques: Infection, Expressing, Control, Selection, Western Blot

Journal: Nature communications

Article Title: ARF triggers senescence in Brca2-deficient cells by altering the spectrum of p53 transcriptional targets.

doi: 10.1038/ncomms3697

Figure Lengend Snippet: Figure 5 | ARF inhibition rescues senescence and proliferation arrest induced by BRCA2 or RAD51 inactivation in mouse and human cells. (a) Early- passage MEFs established from Brca2F/ p53 þ / þ embryos were infected three times at 12-h intervals with retroviruses expressing self-deleting Cre recombinase ( þ Cre) or control pBabe empty vector ( Cre), together with retroviruses expressing ARF or GFP control shRNA, followed by selection with puromycin for 48 h. Cell extracts were prepared 6 days after the first infection and were analysed using western blotting. SMC1 was used as a loading control. (b) Quantification of the SA-b-gal staining of cells treated as in a. Error bars represent s.d. of two independent experiments. The statistical significance of the b-gal response to concomitant Brca2 deletion and ARF depletion was evaluated using an unpaired two-tailed t-test. ARF-1sh and ARF-2sh are two independent shRNAs against mouse ARF. (c) Human MRC5 cells were infected with lentiviruses expressing indicated shRNAs, followed by selection with puromycin for 48 h. Cell extracts were prepared 6 days after infection and were analysed using western blotting. SMC1, GAPDH and tubulin were used as loading controls. (d) Quantification of the SA-b-gal staining of cells treated as in c. Error bars represent s.d. of two independent experiments. The statistical significance of the b-gal response to concomitant BRCA2 and ARF depletion was evaluated using an unpaired two-tailed t-test. (e) Early- passage MEFs established from wild-type, p53 / or Arf / embryos were treated with GFP control or RAD51 shRNAs. Extracts were prepared from cells collected 6 days after infection and immunoblotted as indicated. Tubulin was used as a loading control. (f) Quantification of the SA-b-gal staining of cells treated as in (e). Error bars represent s.d. of two independent experiments. The statistical significance of the b-gal response to RAD51 depletion in the three types of MEFs was evaluated using an unpaired two-tailed t-test. ARF-1sh and ARF-2sh are two independent shRNAs against human ARF. (g) Quantification of the DSBs frequency in metaphase spreads from cells treated as in e. (h) Cell proliferation assays for cells treated as in e. Error bars represent s.d. of three independent experiments.

Article Snippet: The following antibodies were used in immunoblotting: rabbit polyclonal antisera raised against ATM (Sigma Aldrich), H2AX (Calbiochem), SMC1 (Bethyl Laboratories), CHK1 (Cell Signalling Technology), phosphorylated CHK1 (Ser 345, Cell Signalling Technology), phosphorylated CHK2 (Thr 68, Cell Signalling Technology), RAD51 (Santa Cruz Biotechnology), p14 (Abcam), p16 (Santa Cruz Biotechnology), p53 (Santa Cruz Biotechnology), ERK (Cell Signalling Technology), full-length PARP (9542, Cell Signalling Technology), cleaved PARP Asp214 (9541, Cell Signalling Technology), phospho-p53 Ser15 (Cell Signalling Technology), E2F1 (Santa Cruz Biotechnology), DUSP4 (Santa Cruz Biotechnology), DUSP7 (Abcam) and human histone H3 (a gift from A. Verreault); mouse monoclonal antibodies raised against BRCA2 (OP95, Calbiochem), CHK2/Cds1 (Upstate), P-ERK (Cell Signalling Technology), phosphorylated ATM (Cell Signalling Technology), GAPDH (Novus Biologicals), PCNA (Santa Cruz Biotechnology) and a-tubulin68 (Cancer Research UK Monoclonal Antibody Service); rat monoclonal antibody against p19 (Novus); goat polyclonal antibody raised against ATR (Santa Cruz Biotechnology) and sheep polyclonal antibody raised against BRCA2 (ref. 69).

Techniques: Inhibition, Infection, Expressing, Control, Plasmid Preparation, shRNA, Selection, Western Blot, Staining, Two Tailed Test

Journal: Journal of Biological Chemistry

Article Title: Independent Roles for Nibrin and Mre11-Rad50 in the Activation and Function of Atm

doi: 10.1074/jbc.m404294200

Figure Lengend Snippet: FIG. 6. Phosphorylation of Smc1 following irradiation in NBS cells expressing nibrin mutants. NBS-ILB1 cells stably transduced with the pLXIN vector alone, wild type nibrin (NBS1), or the nibrin mutants Nb652, NbFR5, and S278A/S343A were unirradiated or ex- posed to 3 Gy of ionizing radiation, and total cellular lysates were prepared after 60 min. As controls, GM637 normal fibroblasts (control), AT3BI A-T fibroblasts (A-T), and parental NBS-ILB1 cells were in- cluded. 20 g of protein/lane was separated on 3–8% SDS-polyacryl- amide gels and Western-blotted (WB). Immunoblots were probed with a polyclonal antibody specific for the phospho-Ser-957 (P-S957) residue of Smc1, and then were stripped and reprobed with a polyclonal anti-Smc1 antibody. Quantitation of Ser-957 phosphorylation in individual cell lines relative to total Smc1 protein was performed by densitometry and is shown below each lane.

Article Snippet: Primary antibodies to Smc1 included a goat polyclonal antisera (Santa Cruz Biotechnology, Santa Cruz, CA) and rabbit polyclonal antisera specific for the phosphorylated Ser-957 residue, kindly provided by Michael Kastan.

Techniques: Phospho-proteomics, Irradiation, Expressing, Stable Transfection, Transduction, Plasmid Preparation, Control, Western Blot, Residue, Quantitation Assay

Journal: iScience

Article Title: Regulation of DNA damage response by trimeric G-proteins

doi: 10.1016/j.isci.2023.105973

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti-SMC , Cell Signaling Technology , 8E6 (Clone); Cat# 6892, RRID: AB_10828353.

Techniques: Recombinant, Electron Microscopy, Labeling, Magnetic Beads, Protease Inhibitor, Membrane, CRISPR, Plasmid Preparation, Expressing, Software

Journal: Scientific Reports

Article Title: PROTAC-mediated conditional degradation of the WRN helicase as a potential strategy for selective killing of cancer cells with microsatellite instability

doi: 10.1038/s41598-024-71160-5

Figure Lengend Snippet: WRN degradation in MSI but not MSS, cells causes checkpoint activation. ( A ) Representative SDS-PAGE and western blot analysis of lysates from HCT-116 clone 24 treated with 0.3 μM AGB-1 for 0 h, 2 h, 4 h, 8 h or 24 h and blotted with WRN, pSMC1, total SMC1, pCHK2, total CHK2 and GAPDH, as a loading control. Blots from one of three biological repeats are shown. Cis-AGB-1 (0.3 μM) and DMSO (0.1%) treatments for 24 h were used as negative controls for WRN degradation. Cells treated with 5 grays (Gy) of ionising radiation (IR) were used as a positive control for SMC1 and CHK2 phosphorylation. Quantification (mean ± SEM) from all three repeats ( n = 3) are shown alongside with pCHK2: total CHK2 (left) and pSMC1: total SMC1 (right). ( B ), ( C ), ( D ) same as in ( A ) for HCT-116 clone 44, SW620 clone 1 and SW620 clone 17, respectively. Statistical significance was analysed through a one-way ANOVA with a Dunnett post-test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Phospho SMC1 (S966) , Rabbit , 1:5000 , Bethyl , A300-050A.

Techniques: Activation Assay, SDS Page, Western Blot, Control, Positive Control, Phospho-proteomics

Journal: Scientific Reports

Article Title: PROTAC-mediated conditional degradation of the WRN helicase as a potential strategy for selective killing of cancer cells with microsatellite instability

doi: 10.1038/s41598-024-71160-5

Figure Lengend Snippet: List of primary antibodies used for western blots in this project.

Article Snippet: Phospho SMC1 (S966) , Rabbit , 1:5000 , Bethyl , A300-050A.

Techniques: Western Blot

Journal:

Article Title: Phosphorylation of Chk1 by ATR Is Antagonized by a Chk1-Regulated Protein Phosphatase 2A Circuit †

doi: 10.1128/MCB.00447-06

Figure Lengend Snippet: Gö6976 does not induce global phosphorylation of ATR targets or γH2AX foci. HeLa cells were untreated (control) or were incubated with 20 mM HU (2 h), 20 μM VP-16 (4 h), 1 μM Gö6976 (1 h), or 1 μM UCN-01 (2 h). Cell lysates were resolved by SDS-PAGE and subjected to Western blotting with antibodies that recognize phosphorylated substrates of ATM/ATR (A) or phosphorylated Brca1 (B). β-Catenin antibodies were used to verify equal loading. (C) HeLa cells were treated as in panels A and B, except that cells were incubated with Gö6976 for 1 h, 2 h, 3 h, and 6 h before harvest. Samples were analyzed by Western blotting for pS1981-ATM, pS966-Smc1, pS345-Chk1, and Chk1. (D) HeLa cells were treated as in panel C and analyzed by indirect immunofluorescence for γH2AX foci.

Article Snippet: Other primary antibodies used were anti-Cdc25A (Ab-3; Neomarkers), anti-phospho Smc1 S966 (Novus Biologicals), anti-phospho Brca1 S1423 (Chemicon), anti-PP1 (Upstate), anti-PP2A (Upstate), anti-PP4 (recognizes PP2A and PP4) (Abcam), anti-PP6 (Exalpha Biologicals), anti-β-catenin (BD Transduction), anti-γH2AX (Upstate), anti-ATR (Oncogene), and anti-p1981-ATM (Rockland Biochemicals).

Techniques: Incubation, SDS Page, Western Blot, Immunofluorescence