Journal: The Journal of Cell Biology

Article Title: The HINT1 tumor suppressor regulates both γ-H2AX and ATM in response to DNA damage

doi: 10.1083/jcb.200711150

Figure Lengend Snippet: The HINT1 protein is recruited to IRIF and associates with γ-H2AX. (A) Hint1 −/− MEFs were transiently transfected with FLAG-Hint1, grown on coverslips, and treated with 4 Gy of IR. Cells were then fixed after the indicated times and stained with an anti-FLAG antibody (green) together with an anti–γ-H2AX antibody (red) and counterstained for the nucleus with DAPI. Adjacent cells not expressing FLAG-Hint1 were used as an internal negative control. Repeat studies gave similar results. (B) The kinetics of HINT1 and γ-H2AX nuclear foci formation before and after IR treatment. For each genotype and time point, the number of foci per cell was analyzed in 20 images (error bars represent standard deviation). (C) Hint1 −/− MEFs were transiently transfected with FLAG-Hint1 or the control vector (p3xFLAG). 48 h later cells were treated with or without 4 Gy of IR, and whole cell lysates were prepared for immunoprecipitation using the FLAG antibody. Immunoblots were performed using either a FLAG antibody, a γ-H2AX antibody, or an ATM antibody. Repeat studies gave similar results. *, P < 0.05 compared with untreated cells.

Article Snippet: The ATM and TIP60 antibodies were purchased from Santa Cruz Biotechnology, Inc.; histone H2A and γ-H2AX antibodies from Millipore; ac-H2A(K5) antibody from Abcam; p-ATM-S1981, p-Chk1-S317, p-Chk2-T68, p-p53-S15, and p21 antibodies from Cell Signaling Technology; acetyllysine and Rad50 antibodies from Millipore; actin, FLAG, and HA antibodies from Sigma-Aldrich; HRP-conjugated anti–mouse IgG or anti–rabbit IgG secondary antibodies from GE Healthcare; and FITC-conjugated anti–rabbit IgG, Texas red–conjugated anti–rabbit IgG, and Texas red–conjugated anti–mouse IgG secondary antibodies from Vector Laboratories.

Techniques: Transfection, Staining, Expressing, Negative Control, Standard Deviation, Control, Plasmid Preparation, Immunoprecipitation, Western Blot

Journal: The Journal of Cell Biology

Article Title: The HINT1 tumor suppressor regulates both γ-H2AX and ATM in response to DNA damage

doi: 10.1083/jcb.200711150

Figure Lengend Snippet: Loss of HINT1 impairs the acetylation and activation of ATM. (A) Hint1 MEFs were untreated or treated with 4 Gy of IR, and whole cell lysates were collected after 10 min. Immunoprecipitates were obtained using an anti-ATM antibody and resolved on SDS-PAGE. Immunoblots were then performed using either an ATM antibody or an acetyllysine antibody. (B) Hint1 +/+, +/−, and −/− MEFs were untreated (0) or treated with 4 Gy of IR. After the indicted times, cell lysates were collected and immunoblot analyses done for the levels of p-ATM-S1981 and ATM. Actin was used as an internal loading control. (C) Hint1 +/+, +/−, and −/− MEFs were untreated or treated with 4 Gy of IR and fixed after the indicated times. Cells were then stained with an antibody to p-ATM-S1981 (red) and counterstained for the nucleus with DAPI (blue). Bar, 10 μm. (D and E) Cell lysates from Hint1 MEFs untreated (0) or treated with 4 Gy of IR (D) or 100 nM bleomycin (E) were collected at the indicated times, and immunoblot analyses were done for the levels of p-Chk1-S317, p-Chk2-T68, and p-p53-S15. A repeated experiment yielded similar results.

Article Snippet: The ATM and TIP60 antibodies were purchased from Santa Cruz Biotechnology, Inc.; histone H2A and γ-H2AX antibodies from Millipore; ac-H2A(K5) antibody from Abcam; p-ATM-S1981, p-Chk1-S317, p-Chk2-T68, p-p53-S15, and p21 antibodies from Cell Signaling Technology; acetyllysine and Rad50 antibodies from Millipore; actin, FLAG, and HA antibodies from Sigma-Aldrich; HRP-conjugated anti–mouse IgG or anti–rabbit IgG secondary antibodies from GE Healthcare; and FITC-conjugated anti–rabbit IgG, Texas red–conjugated anti–rabbit IgG, and Texas red–conjugated anti–mouse IgG secondary antibodies from Vector Laboratories.

Techniques: Activation Assay, SDS Page, Western Blot, Control, Staining

Journal: Genes & Development

Article Title: ATR checkpoint kinase and CRL1 βTRCP collaborate to degrade ASF1a and thus repress genes overlapping with clusters of stalled replication forks

doi: 10.1101/gad.239194.114

Figure Lengend Snippet: DOX induces ATR-CRL1 βTRCP -dependent ASF1a degradation. ( A ) Immunoblots of cell lysates. ASF1a, but not ASF1b or CAF1, was decreased by DOX for 20 h, and this decrease was relieved by 5 mM caffeine added to cells from −4 h relative to DOX until the time of harvest. ACTIN was used as a loading control for the immunoblots. ( B , left panel) ASF1a/b examined in cells transfected with siGL2, siATR, or siATM for 24 h and then treated with DOX for 20 h under continued exposure to siRNAs. ( Right panel) Knockdown of endogenous ATR or ATM by siRNA was confirmed by immunoprecipitation (IP) and Western blot (WB). The IgG amount in each lane served as the loading control for the immunoprecipitates, and ACTIN shows equal amounts of whole -cell extracts (WCEs). ( C ) Cells treated with DOX for 20 h were exposed to 10 μM MG132 for 4 h before harvest. Endogenous ASF1a and ACTIN were immunoblotted. ( D ) Cells transfected with two different siRNAs to βTRCP for 24 h and a siRNA to Cullin1 for 48 h before DOX treatment for 20 h with continued incubation with siRNA and subjected to Western blotting for the indicated proteins and ACTIN (loading control). ( E ) After transfection of siGL2 or siATR (24 h) or exposure to 5 mM caffeine (4 h), cells were treated with DOX or DMSO for 20 h with continued incubation with siRNA or caffeine. Cells were harvested after exposure to 10 μM MG132 for 4 h to stabilize the ASF1a. Immunoprecipitates ( top four panels) or input whole-cell extracts ( bottom three panels) were immunoblotted for the indicated proteins. IgG served as a loading control for immunoprecipitates, and Cullin1 served as a loading control for whole-cell extracts. ( F ) An experiment similar to E , except immunoprecipitation done with anti-βTRCP antibody.

Article Snippet: Antibodies against each protein were purchased from the following companies: CHK1, Cyclin A, Cyclin E, CUL1, and CAF1 from Santa Cruz Biotechnology; p-CHK1(Ser345), ASF1a, ASF1b, and βTRCP from Cell Signaling Technology; ATR from Affinity Bioreagent; ATM from Novus Biological; and BRCA1 from Milipore.

Techniques: Western Blot, Control, Transfection, Knockdown, Immunoprecipitation, Incubation

Journal: Genes & Development

Article Title: ATR checkpoint kinase and CRL1 βTRCP collaborate to degrade ASF1a and thus repress genes overlapping with clusters of stalled replication forks

doi: 10.1101/gad.239194.114

Figure Lengend Snippet: The ATR-dependent checkpoint pathway is required for the transcriptional repression of genes overlapping with clusters of stalled forks. ( A , B ) Histone H3 ( A ) and RPA ( B ) ChIP assay for three genes overlapping with stalled forks and a control distal gene, WNT2 . The rest are as in . (DOX-CF) DOX-treated cells exposed to caffeine for 24 h before harvesting. ( C ) RNA polymerase II (pol II) ChIP assay at the promoters of three genes overlapping with stalled forks and a control distal gene, WNT2 . (DOX-CF) DOX-treated cells exposed to caffeine for 24 h before harvesting. ( D ) Cells depleted of ATR or ATM by siRNA were treated with 1.5 μM DOX or DMSO (Con). The relative mRNA expression of three overlapped genes and a control gene, p21 , was analyzed by qRT–PCR as in the Materials and Methods. Student’s t -test analysis, (*) P < 0.05.

Article Snippet: Antibodies against each protein were purchased from the following companies: CHK1, Cyclin A, Cyclin E, CUL1, and CAF1 from Santa Cruz Biotechnology; p-CHK1(Ser345), ASF1a, ASF1b, and βTRCP from Cell Signaling Technology; ATR from Affinity Bioreagent; ATM from Novus Biological; and BRCA1 from Milipore.

Techniques: Control, Expressing, Quantitative RT-PCR

Journal: The American journal of pathology

Article Title: Up-Regulation of DNA Damage Response Signaling in Autosomal Dominant Polycystic Kidney Disease.

doi: 10.1016/j.ajpath.2021.01.011

Figure Lengend Snippet: Figure 2 Immunohistochemistry for DNA damage marker, g-H2A histone family member X (H2AX), and DNA damage response signaling kinases, phos- phorylated ataxia telangiectasia and Rad3 related (p-ATR), and phosphorylated AT mutated (p-ATM), in human autosomal dominant polycystic kidney disease (ADPKD) kidney compared with normal human kidney. A: Representative micrographs. For each protein, the same micrograph section from the Zyagen (HP-901; San Diego, CA) sample is shown for normal kidney and the same micrograph section from ADPKD patient P1 is shown for ADPKD (both minimally cystic and cystic). B: Quantification of g-H2AXepositive staining. C: Quantification of p-ATRepositive staining. D: Quantification of p-ATMepositive staining. n Z 8 human ADPKD kidney (BeD); n Z 3 normal human kidney (BeD); n Z 3 to 4 per group (BeD). *P < 0.05 versus normal kidney (analysis of variance, followed by post-hoc analysis with the Tukey-Kramer honestly significant difference test). Scale bars Z 50 mm.

Article Snippet: Primary antibodies used were as follows: i) antiATR (phospho T1989; 1:1000, ab227851; Abcam), ii) anti- ajp.amjpathol.org - The American Journal of Pathology ATR (1:1000, ab2905; Abcam), iii) anti-ATM (phospho S1981; 1:1000, ab81292; Abcam), and iv) anti-ATM (1:1000, NB100-104; Novus Biologicals, Littleton, CO).

Techniques: Immunohistochemistry, Marker, Staining

Journal: The American journal of pathology

Article Title: Up-Regulation of DNA Damage Response Signaling in Autosomal Dominant Polycystic Kidney Disease.

doi: 10.1016/j.ajpath.2021.01.011

Figure Lengend Snippet: Figure 3 Expression of DNA damage marker, g-H2A histone family member X (H2AX), and DNA damage response kinases, phosphorylated (p) and total ataxia telangiectasia and Rad3-related (ATR) and phosphorylated and total AT mutated (ATM) in unstimulated human autosomal dominant polycystic kidney disease tubular cells (WT 9-7 and WT 9-12) compared with normal kidney tubular cells (HK-2) by immunocytochemistry and Western blot analysis. A: Representative images of positive g-H2AX staining. B: Number of g-H2AX foci per cell for each cell type. C: Representative blots of p-ATR and total ATR expression. D: Representative blots of p-ATM and total ATM expression. E: Fold change in p-ATR expression in WT 9-7 and 9-12 cells compared with HK-2. F: Fold change in total ATR expression in WT 9-7 and 9-12 cells compared with HK-2. G: Fold change in p-ATM in WT 9-7 and 9-12 cells compared with HK-2. H: Fold change in total ATM in WT 9-7 and 9-12 cells compared with HK-2. Data presented as means SD (B and EeH). n Z 3 for each cell type (B); n Z 9 for each cell type (EeH). *P < 0.05 versus HK-2 (analysis of variance, followed by post-hoc analysis with the Tukey-Kramer honestly significant difference) test. Images obtained with a 20 objective (A).

Article Snippet: Primary antibodies used were as follows: i) antiATR (phospho T1989; 1:1000, ab227851; Abcam), ii) anti- ajp.amjpathol.org - The American Journal of Pathology ATR (1:1000, ab2905; Abcam), iii) anti-ATM (phospho S1981; 1:1000, ab81292; Abcam), and iv) anti-ATM (1:1000, NB100-104; Novus Biologicals, Littleton, CO).

Techniques: Expressing, Marker, Immunocytochemistry, Western Blot, Staining

Journal: The American journal of pathology

Article Title: Up-Regulation of DNA Damage Response Signaling in Autosomal Dominant Polycystic Kidney Disease.

doi: 10.1016/j.ajpath.2021.01.011

Figure Lengend Snippet: Figure 8 Relative expression of DNA damage response genes, Atr (A), Atm (B), Chek1 (C), and Chek2 (D), in male wild-type (WT) and Pkd1RC/RC mice aged 1, 3, and 6 months, as measured by quantitative real-time PCR. Relative fold changes in gene expression were quantified using the 2DDC T method, relative to age- matched wild-type average and Gapdh and Actb as reference genes. Data presented as means SD (AeD). n Z 4 to 6 per group per time point (AeD). *P < 0.05 versus age-matched wild-type mice (independent t-test).

Article Snippet: Primary antibodies used were as follows: i) antiATR (phospho T1989; 1:1000, ab227851; Abcam), ii) anti- ajp.amjpathol.org - The American Journal of Pathology ATR (1:1000, ab2905; Abcam), iii) anti-ATM (phospho S1981; 1:1000, ab81292; Abcam), and iv) anti-ATM (1:1000, NB100-104; Novus Biologicals, Littleton, CO).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Gene Expression

Journal: Oncogene

Article Title: Transgenic expression of E2F3a causes DNA damage leading to ATM-dependent apoptosis.

doi: 10.1038/onc.2008.138

Figure Lengend Snippet: Figure 2 Overexpression of E2F3a activates ATM. (a) Western blot analysis was performed on epidermal lysates from nontrans- genic (lanes 1,2, 5 and 6) or K5 E2F3a transgenic (lanes 3, 4, 7 and 8) mice that were either wild-type (lanes 1–4) or null (lanes 5–8) for Atm. Antibodies specific for E2F3, phospho-ATM S1981 and b-tubulin were used as indicated. (b) Skin sections from wild-type, K5 E2F3a, Atm/ and K5 E2F3a Atm/ mice were immunohis- tochemically stained for the phosphorylated form of p53 (serine 18 in mouse). Positively stained epidermal keratinocytes were identi- fied microscopically and the average number per 10 mm of linear epidermis from at least three mice per group is presented. (c) Western blot analysis was performed on lysates from primary NHFs (lanes 1–3) or primary fibroblasts from an AT patient (lanes 4–6) infected with AdCMV empty vector (lanes 1 and 4), treated with etoposide as a positive control for DNA damage (lanes 2 and 5) or infected with AdE2F3a (lanes 3 and 6). Antibodies specific for E2F3, phospho-p53 S15, phospho-ATM S1981 and b-tubulin were used as indicated. ATM, ataxia telangiectasia mutated; NHFs, normal human fibroblasts. *indicates statistical significance at Po0.05.

Article Snippet: The following antibodies were used to detect the indicated protein: phospho-ATM S1981 (Rockland, Gilbertsville, PA, USA), E2F3 (Santa Cruz Biotechnology, C-18), b-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA, USA, H-235), phospho-p53 S15 (Cell Signaling Technology), b-actin (Santa Cruz Biotechnology, H-2350).

Techniques: Over Expression, Western Blot, Transgenic Assay, Staining, Infection, Plasmid Preparation, Positive Control