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: Cell Reports Medicine

Article Title: A kinome-wide CRISPR screen identifies CK1α as a target to overcome enzalutamide resistance of prostate cancer

doi: 10.1016/j.xcrm.2023.101015

Figure Lengend Snippet:

Article Snippet: ATM gRNA2 , Doench et al. , Addgene Cat# 77531.

Techniques: Magnetic Beads, Virus, Recombinant, Mutagenesis, Plasmid Preparation, CRISPR, Knock-Out, Software