Journal: Arthritis and rheumatism

Article Title: Defective DNA double-strand break repair in pediatric systemic lupus erythematosus.

doi: 10.1002/art.33334

Figure Lengend Snippet: Figure 4. Kinetics of phosphorylation of structural maintenance of chromosomes protein 1 (SMC1). A, Sixteen lymphoblastoid cell lines from patients with systemic lupus erythematosus (SLE) were irradiated with 10 Gy and harvested following incubation periods of 0 minutes, 15 minutes, 30 minutes, 1 hour, 4 hours, and 24 hours. Nuclear lysates were used in immunoblot analysis. WT (CHOC6, Paris1) control cell lines and RS (AT204LA, RS13) control cell lines were used in each experiment. Asterisks indicate SLE cell lines with slower resolution of SMC1 phosphorylation. Lower rows in each panel show loading controls of native SMC1. B, Shown is quantification of SMC1 phosphorylation. WT control cell lines 1 and 2 reach peak phosphorylation between 30 minutes and 1 hour postirradiation, with near-complete resolution of phosphorylation by 24 hours. Dots indicate where phosphorylation was aberrant. Lig 4 indicates DNA ligase IV (Lig 4)–deficient cell line (RS13). See Figure 1 for other definitions.

Article Snippet: For visualizing Fanconi protein D2 monoubiquitination products or SMC1 phosphorylation, a 7.5% SDSPAGE gel was electrophoresed; the immunoblot was incubated overnight with either FANCD2 antibody or SMC1 [p Ser966] antibody (both from Novus Biologicals); secondary antibody was detected with an HRP conjugate.

Techniques: Phospho-proteomics, Irradiation, Incubation, Western Blot, Control

Journal: Arthritis and rheumatism

Article Title: Defective DNA double-strand break repair in pediatric systemic lupus erythematosus.

doi: 10.1002/art.33334

Figure Lengend Snippet: Figure 5. A, Irradiation-induced 53BP1 foci formation. Retention of 53BP1 at double-strand break sites measures the integrity of the ubiquitin ligase cascade. Formation of 53BP1 foci was normal in all tested lymphoblastoid cell lines from patients with systemic lupus erythematosus (SLE) at 1 hour after irradiation with 3 Gy. WT (Paris1) and RS66 (RNF168-deficient) cell lines were used as positive and negative controls, respectively. H2AX foci (not shown) were used as irradiation controls and were positive for each cell line shown. Original magnification 40. B, Bromodeoxyuridine (BrdU) incorporation after irradiation with 10 Gy. All 8 SLE cell lines tested were within a normal range. Mean SD BrdU incorporation was 16.97 6.54% in a WT cell line (NAT9) and 79.26 14.52% in an A-T cell line (AT204LA). C, Monoubiquitination of Fanconi protein D2 (FANCD2). Both the ubiquitinated (FANCD2-L) and nonubiquitinated (FANCD2-S) forms were present in the protein extracts from all SLE cell lines. Nuclear extract from a Fanconi protein D2–deficient cell line (PD20.L) was used as a negative control; structural maintenance of chromosomes protein 1 (SMC1) was used as a loading control. D, Immunoblot showing ATM protein levels. ATM protein levels were within normal limits for the 8 SLE cell lines tested. The SMC1 loading control shows that SLE cell line 71 was slightly underloaded. See Figure 1 for other definitions.

Article Snippet: For visualizing Fanconi protein D2 monoubiquitination products or SMC1 phosphorylation, a 7.5% SDSPAGE gel was electrophoresed; the immunoblot was incubated overnight with either FANCD2 antibody or SMC1 [p Ser966] antibody (both from Novus Biologicals); secondary antibody was detected with an HRP conjugate.

Techniques: Irradiation, Ubiquitin Proteomics, BrdU Incorporation Assay, Negative Control, Control, Western Blot

Journal: Arthritis and rheumatism

Article Title: Defective DNA double-strand break repair in pediatric systemic lupus erythematosus.

doi: 10.1002/art.33334

Figure Lengend Snippet: Figure 6. Non-homologous DNA end joining (NHEJ) pathway. A, NHEJ assay. Ten micrograms of nuclear lysate from lymphoblastoid cell lines from patients with systemic lupus erythematosus (SLE) was added to 250 ng of Sma I–digested (SmaI[]) pUC19 DNA. Nuclear lysates from all SLE cell lines and a wild-type (WT) control cell line (Paris1) efficiently ligated Sma I–digested plasmid, forming higher-order multimers (upper right). Exclusion of MgCl2 (WT[]MgCl) served as a negative control (see Materials and Methods). Quantification of ligation efficiency was calculated using the dimer and a representative multimer band (asterisk). A linear form of the plasmid (Uncut pUC19) was used as the loading control. B, Immunoblot analysis of 7 core proteins involved in NHEJ. Fifty micrograms of whole cell extract isolated from each SLE lymphoblastoid cell line was used to immunoblot for DNA-dependent protein kinase, catalytic subunit (DNA-PKcs), Ku70, Ku80, Artemis, XLF/Cernunnos (XLF), DNA ligase IV (Lig 4), and XRCC4. Beta-actin or structural maintenance of chromosomes protein 1 (SMC1) was used as a loading control and is shown below the corresponding immunoblots. All 7 core proteins were present in the 16 SLE cell line extracts tested. SLE 64 and SLE 73 were retested for Ku70 and Ku80; SLE 68 was retested for XRCC4 and Lig 4. All cell lines were normal (not all data are shown).

Article Snippet: For visualizing Fanconi protein D2 monoubiquitination products or SMC1 phosphorylation, a 7.5% SDSPAGE gel was electrophoresed; the immunoblot was incubated overnight with either FANCD2 antibody or SMC1 [p Ser966] antibody (both from Novus Biologicals); secondary antibody was detected with an HRP conjugate.

Techniques: NHEJ Assay, Control, Plasmid Preparation, Negative Control, Ligation, Western Blot, Isolation

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: 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: ATM-dependent CHK2 Activation Induced by Anticancer Agent, Irofulven

doi: 10.1074/jbc.m400015200

Figure Lengend Snippet: FIG. 7. ATM and CHK2 target proteins activated by irofulven. A and B, human ovarian cancer cell lines A2780, CAOV3, SKOV3, and OVCAR3 were treated with 1 IC50 concentration of irofulven (A); and human normal fibroblast GM00637 and AT fibroblast GM05849 were treated with 8 M of irofulven (B) for 1 h followed by additional 24 h of incubation. Western blot analyses were performed with antibodies against phosphorylated NBS1 on Ser343, NBS1, phosphorylated SMC1 on Ser957, SMC1, and phosphorylated p53 on Ser15. The nonspecific band was indicated as n.s. C and D, A2780 and CAOV3 cells (C); and human colon cancer cell line HCT116 and its CHK2 knockout subline (D) were treated with 1 IC50 concentration of irofulven for 1 h followed by additional 24 h of incubation, Western blots were performed with antibodies against phosphorylated p53 on Ser20, p53, and actin.

Article Snippet: Monoclonal antibody against PARP and polyclonal antibodies against ATR, SMC1, NBS1, CDC25A, p53, and phosphorylated p53 on Ser15 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Concentration Assay, Incubation, Western Blot, Knock-Out

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Overexpression of the cohesin-core subunit SMC1A contributes to colorectal cancer development.

doi: 10.1186/s13046-019-1116-0

Figure Lengend Snippet: Fig. 1 Comprensive analysis of SMC1A-cohesin gene at different steps during CRC development. OncoScan was used to obtain both CNVs and LOH in mucosa (n = 16), early adenoma (n = 16) and carcinoma (n = 16) samples. a CNVs profile in mucosa. b CNVs profile in adenomas. c CNVs profile in carcinoma showing the gain of whole chromosomes 7, 13, and X and the loss of chromosome 18. d Mutational screening in colorectal early adenomas and carcinoma allowed us to identify twenty-five SMC1A mutations (16 mutations in carcinomas and 9 in adenomas, see Table 2). Example of representative SMC1A sequencing is reported showing the nucleotide change c.G1966A (leading to p.A656T amino acid change) identified in patient 6. e Diagram of the SMC1A protein with mutations identified in carcinomas (above) and adenomas (below). The protein length is not in scale. f Percentage of subjects (n = 66) analyzed by SMC1A immunohistochemistry and showing strong, moderate and weak intensity. g Examples of representative immunohistochemistry results are shown. It is evident that the expression of SMC1A protein increased during cancer development

Article Snippet: SMC1A cDNA mutagenesis and cell transfection Site-directed mutagenesis of the SMC1A cDNA (OriGene) was performed with QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer’s instructions.

Techniques: Sequencing, Immunohistochemistry, Expressing

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Overexpression of the cohesin-core subunit SMC1A contributes to colorectal cancer development.

doi: 10.1186/s13046-019-1116-0

Figure Lengend Snippet: Fig. 2 Effects of SMC1A mutation and overexpression in vivo. a HCT116 cells, a colorectal cancer cell line with stable karyotype, were stably transfected with both SMC1A wild-type gene and SMC1A c.A2027G mutation. Western blot shows a more marked expression in HCT116 SMC1A wild-type (2) and HCT116 SMC1A c.A2027G (3) when compared with HCT116 cells (1). An antibody against Tubulin was used as loading control. b The effects of HCT116, HCT116 SMC1A wild-type and HCT116 SMC1A c.A2027G cells were analyzed in vivo using an immunocompromised mouse model. Time-dependent analysis shows that HCT116 SMC1A wild-type and HCT116 SMC1A c.A2027G require 11 and 13 days to form tumors. In contrast, the development of tumors peaked after 18 days with HCT116 cells. c Representative images of tumors formed in the mice with HCT116 cells. d Tumors induced by HCT116 SMC1A wild-type. e Tumors formed in the mice in which HCT116 SMC1A c.A2027G cells were implanted. f Change in tumor weight. g Difference in tumor volume after subcutaneous cell inoculation. h Example of representative histopathological examination performed with hematoxylin and eosin staining. Enlargement 500x. *p < 0.05

Article Snippet: SMC1A cDNA mutagenesis and cell transfection Site-directed mutagenesis of the SMC1A cDNA (OriGene) was performed with QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer’s instructions.

Techniques: Mutagenesis, Over Expression, In Vivo, Stable Transfection, Transfection, Western Blot, Expressing, Control, Staining

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Overexpression of the cohesin-core subunit SMC1A contributes to colorectal cancer development.

doi: 10.1186/s13046-019-1116-0

Figure Lengend Snippet: Fig. 3 Gene expression profile in induced tumors. a RNA-seq data shows that HCT116 SMC1A wild-type induced tumors display 401 up- and 343 down-regulated genes, whereas HCT116 SMC1A c.A2027G-induced tumors show 486 up- and 256 down-regulated genes. b Venn diagram showing that HCT116 SMC1A wild-type and HCT116 SMC1A c.A2027G share sixty-eight dysregulated genes. c Heatmap of sixty-eight dysregulated genes showing that the transcriptional effects were very small, with fold changes ranging from + 0.8 to −0.8

Article Snippet: SMC1A cDNA mutagenesis and cell transfection Site-directed mutagenesis of the SMC1A cDNA (OriGene) was performed with QuikChange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer’s instructions.

Techniques: Gene Expression, RNA Sequencing

Journal: Nucleic Acids Research

Article Title: Protein phosphatase 1 and phosphatase 1 nuclear targeting subunit-dependent regulation of DNA-dependent protein kinase and non-homologous end joining

doi: 10.1093/nar/gkx686

Figure Lengend Snippet: PP1 functions in NHEJ via regulation of DNA-PKcs. ( A ) The linearized NHEJ template was repaired in Xenopus egg extracts, as in Figure . The extracts were depleted of PP1 (using the PP-binding motif of PNUTS, as in Figure ), or treated with a DNA-PKcs inhibitor (NU7441, 20 uM), as indicated. The repair efficiency, measured by colony formation, is shown. Extract samples were analyzed by immunoblotting for DNA-PKcs phospho-S2056, PP1γ and H2B. The experiment was performed at least three times, and the results are shown as the mean values and standard deviations. Statistical significance was analyzed using an unpaired two-tailed Student's t -test. ( B ) Xenopus egg extracts with or without PP1 depletion (using the PP-binding motif of PNUTS) were treated with 20 ng/μl of (dA-dT) 70 as indicated. Immunoblots of DNA-PKcs phospho-S2056, DNA-PKcs phospho-T2609, DNA-PKcs, PP1β, PP1γ and H2B are shown. ( C ) In vitro DNA-PKcs kinase assay was performed using XRCC4 as substrate, as described in Materials and Methods. The autoradiography and Coomassie stain of XRCC4 are shown. ( D ) Xenopus egg extracts were immunodepleted of PP1β, PP1γ or both. The extracts were treated with (dA-dT) 70 for 30 min, and analyzed by immunoblotting for DNA-PKcs phospho-S2056, DNA-PKcs, PP1β, PP1γ and H2B. ( E ) HeLa cells were transfected with control or PP1γ siRNA, treated with 10 Gy IR, and incubated as indicated. Immunoblots of DNA-PKcs phospho-S2056, DNA-PKcs, Smc1 phospho-S957, PP1γ and H2B are shown. ( F ) Five segments of DNA-PKcs (N, JK, PQR, ABCDE (ABC) and C) were expressed with MBP-tag, and purified on amylose beads, as described in Materials and Methods. The beads were then incubated in HeLa cell lysates, re-isolated, and analyzed by immunoblotting. The ‘Ctr’ sample was a mock pull-down using empty amylose beads. (G–I) Immunoprecipitation of PP1γ ( G ), PP5 ( H ) and PP6 ( I ) was performed in the lysates of HeLa cell with or without doxorubicin treatment. The lysate input, control IP with blank beads, and PP1γ IP products were analyzed by immunoblotting for DNA-PKcs, Ku70 and PP1γ, PP5 and PP6, as indicated.

Article Snippet: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were carried out as previously described , using the following antibodies: Artemis (A300-234A), Chk1 phospho-S317 (A304-673A), Ku80 (A302-627A), γ-H2AX (A300-081A), PP1β (A300-905A), PP1γ (A300-906A), PP5 (A300-909A), PP6 (A300-844A), PNUTS (A300-439A) and Smc1 phospho-S957 (A304-147A) antibodies from Bethyl Laboratories (Montgomery, TX, USA); GFP (sc-9996) and Ku70 (sc-56129) antibodies from Santa Cruz Biotechnology (Dallas, TX, USA); ATM phospho-S1981 (ab36810), Chk2 phospho-T387 (ab195783), DNA-PKcs (ab70250), DNA-PKcs phospho-S2056 (ab18192), DNA-PKcs phospho-T2609 (ab18356) and H2B (ab1790-100) antibodies from Abcam (Cambridge, MA, USA); β-actin (#4970), BRCA1 phospho-S1524 (#9009P), Chk1 (#2345), Chk1 phospho-S296 (#2349), Chk2 phospho-T68 (#2661) and Rad52 (#3425P) antibodies from Cell Signaling Technology (Beverly, MA, USA); DNAL IV (GTX100100), and XRCC4 (GTX100094) antibodies from GeneTex (Irvine, CA, USA).

Techniques: Binding Assay, Western Blot, Two Tailed Test, In Vitro, Kinase Assay, Autoradiography, Staining, Transfection, Control, Incubation, Purification, Isolation, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: Protein phosphatase 1 and phosphatase 1 nuclear targeting subunit-dependent regulation of DNA-dependent protein kinase and non-homologous end joining

doi: 10.1093/nar/gkx686

Figure Lengend Snippet: PNUTS mediates the DNA damage response and repair. ( A ) Xenopus egg extracts were supplemented with (dA-dT) 70 (DNA damage), WT or W393A PNUTS, as indicated, and incubated for 30 min. The extract samples were analyzed by immunoblotting for Brca1 phospho-S1524, Chk2 phospho-T387, Smc1 phospho-S957, Chk1 phospho-S317 and Chk1. In the lower panel, the relative intensity of Chk1 phospho-S317/Chk1 was measured using NIH ImageJ, and shown by the mean values and standard deviations. ( B ) HeLa cells were transfected with GFP-PNUTS, and treated with 5 μM doxorubicin for 4 h, as indicated. The cell lysates were analyzed by immunoblotting for ATM phospho-S1981, Chk2 phospho-T68, Chk1 phoshpo-S296, GFP and H2B. ( C ) HeLa cells with or without GFP-PNUTS expression were treated with doxorubicin for 24 h. Cell death was measured by trypan blue excursion. ( D ) HeLa cells were treated with PNUTS siRNA and doxorubicin, as indicated. The cell number of each day was normalized to that of the first day for the relative cell viability. ( E ) HeLa cells with control or PNUTS siRNA were irradiated with 4 Gy IR and incubated as indicated. The cell lysates were analyzed by immunoblotting for γ-H2AX, PNUTS and β-actin. (F, G) HeLa cells treated as in panel E were examined by immunofluorescence for γ-H2AX. The average numbers of γ-H2AX foci were counted ( F ) and representative images of cells stained for γ-H2AX (in red) and DAPI (in blue) are shown ( G ). At least 100 cells were analyzed for γ-H2AX foci in panel F.

Article Snippet: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were carried out as previously described , using the following antibodies: Artemis (A300-234A), Chk1 phospho-S317 (A304-673A), Ku80 (A302-627A), γ-H2AX (A300-081A), PP1β (A300-905A), PP1γ (A300-906A), PP5 (A300-909A), PP6 (A300-844A), PNUTS (A300-439A) and Smc1 phospho-S957 (A304-147A) antibodies from Bethyl Laboratories (Montgomery, TX, USA); GFP (sc-9996) and Ku70 (sc-56129) antibodies from Santa Cruz Biotechnology (Dallas, TX, USA); ATM phospho-S1981 (ab36810), Chk2 phospho-T387 (ab195783), DNA-PKcs (ab70250), DNA-PKcs phospho-S2056 (ab18192), DNA-PKcs phospho-T2609 (ab18356) and H2B (ab1790-100) antibodies from Abcam (Cambridge, MA, USA); β-actin (#4970), BRCA1 phospho-S1524 (#9009P), Chk1 (#2345), Chk1 phospho-S296 (#2349), Chk2 phospho-T68 (#2661) and Rad52 (#3425P) antibodies from Cell Signaling Technology (Beverly, MA, USA); DNAL IV (GTX100100), and XRCC4 (GTX100094) antibodies from GeneTex (Irvine, CA, USA).

Techniques: Incubation, Western Blot, Transfection, Expressing, Control, Irradiation, Immunofluorescence, Staining

Journal: Cell cycle (Georgetown, Tex.)

Article Title: Evidence for hSNM1B/Apollo functioning in the HSP70 mediated DNA damage response.

doi: 10.4161/cc.8.11.8605

Figure Lengend Snippet: Figure 3. Induction of DNA damage does not quantitatively affect the hSN- M1B/HSP70 interaction. HEK293T cells were transiently transfected with a Flag-hSNM1B expression plasmid or the empty vector. Cells were lysed 24 h post transfection following a 1.5 h Bleomycin treatment (25 μg/ml) and immunoprecipitation was performed using antibodies against the Flag-tag. Blots were probed with Flag antibodies to show transient Flag-hSNM1B expression and co-immunoprecipitated HSC70/HSP72 was detected by antibodies against HSP70. Aliquots of the lysates used for IP were analysed with antibodies specifically recognizing SMC1 phosphorylated at serine 957 as a control for the successful Bleomycin treatment and antibodies against total SMC1 to show equal loading.

Article Snippet: Many of the HSP70 proteins have been found to be overexpressed in tumors leading to distinct mechanisms for promoting cell survival.35 For example, increased expression of HSP72 in prostate carcinoma cells confers resistance to a variety of anticancer agents.36 Consequentialy, targeting HSPs is a promising D ow nl oa de d by [ 94 .2 42 .2 28 .1 08 ] at 0 6: 20 1 6 M ar ch 2 01 6 www.landesbioscience.com Cell Cycle 1731 the following primary antibodies: SMC1, actin, HSP70, CHK1, (abcam), CHK1 phospho-serine 317 (Novus Biologicals), SMC1 phospho-serine 957 (Bethyl Laboratories), TRF2 (Imgenex), CBP (Cell Signaling), Myc-tag, Flag-tag (Sigma-Aldrich).

Techniques: Transfection, Expressing, Plasmid Preparation, Immunoprecipitation, FLAG-tag, Control