Journal: Nature cell biology

Article Title: SARS-CoV-2 infection induces DNA damage, through CHK1 degradation and impaired 53BP1 recruitment, and cellular senescence.

doi: 10.1038/s41556-023-01096-x

Figure Lengend Snippet: Fig. 4 | SARS-CoV-2 ORF6 or NSP13 expression is sufficient to cause CHK1 loss. a, Immunofluorescence (IF) images of Huh7 cells expressing Strep-tagged SARS-CoV-2 ORF6 or NSP13 fixed 48 h post-transfection and stained for DDR markers; GFP was used as control; staining with anti-Strep-tag was used to label transfected cells; nuclei were stained with DAPI. Scale bar, 10 μm. b, The histograms show the percentage of CHK1- or RRM2-expressing cells among the transfected ones (Strep-Tag+) as determined in a; n = 3 independent experiments (n = 4 for GFP-expressing cells in RRM2 analysis). The dot plots show the number

Article Snippet: 3 nature portfolio | reporting sum m ary M arch 2021 Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used gH2AX (Ser139) Abcam ab11174 gH2AX (Ser139) Millipore 05-636 53BP1 Bethyl A303-906A 53BP1 Novus NB100-304 ACE2 Abcam ab15348 ATM Abcam ab32420 ATR Santa Cruz sc-1887 Beclin 1 Bethyl A302-566A-T Beta-actin Sigma-Aldrich A2228 BrdU BDbioscience 347580 CD68 Abcam ab125212 CDT1 Cell Signaling #8064 cGAS Cell Signaling #15102 CHK1 Novus NB100-46 CHK1 (2G1D5) Cell Signaling #2360 CHK1 (ST57-09) ThermoFisher MA532180 CHK2 Millipore 05-649 Cleaved Caspase-3 (Asp 175) Cell Signaling 9661 DNA-PK Abcam ab32566 HA-tag Abcam ab236632 Histone H3 Abcam ab10799 Human Cytokeratin 8/18 (EP17/EP30) Dako M3652 ISceI (FL-86) Santa Cruz sc-98269 KAP1 Abcam ab10484 LC3B Sigma-Aldrich L7543 p16 Abcam ab51243 p21 Abcam ab188224 P21 Cell Signaling #2946 P38 MAPK Cell Signaling #9212 P53 Abcam ab1101 p62 Abcam ab240635 pATM (Ser1981) Rockland 200-301-400 pATR (T1989) Abcam ab223258 pCHK1 (S317) Cell Signaling #2344 pCHK2 (Thr68) Cell Signaling #2661 PCNA Bio Rad MCA1558 pDNA-PK (Ser2056) (EPR5670) Abcam ab124918 pKAP1 (S824) Bethyl A300-767A pP38 MAPK (Thr180/Tyr182) Cell Signaling #9211 pP53 (Ser15) Cell Signaling #9284 proSP-C Abcam ab3786 pRPA (S4/S8) Bethyl A300-245A pSTAT1 (Ser 727) Cell Signaling #9177 pSTAT1 (Tyr 701) (58D6) Cell Signaling #9167 RPA Calbiochem NA18-100UG RRM1 Santa Cruz sc-11733 RRM2 Novus NBP1-31661 RRM2 Santa Cruz sc-10844 SARS-CoV2 nucleocapsid Sino Biological 40588-T62 SARS-CoV2 nucleocapsid Sino Biological 40143-R019 SARS-CoV2 nucleocapsid (1A6) ThermoFisher MA5-35941 STAT1 (9H2) Cell Signaling #9176 Strep-tag II epitope Qiagen 34850 Tubulin Sigma-Aldrich T5168 Ubiquitin (P4D1) Santa Cruz sc-8017 Vinculin Sigma-Aldrich V9131 Cy3 D/M Jackson 715-165-150 Cy3 D/R Jackson 711-165-152 Cy3 D/G Jackson 705-165-147 4 nature portfolio | reporting sum m ary M arch 2021 A488 D/M ThermoFisher A21202 A488 D/R ThermoFisher A21206 A488 D/G ThermoFisher A11055 A647 D/M ThermoFisher A31571 A647 D/R ThermoFisher A31573 A647 D/G ThermoFisher A21447 Validation All antibodies were validated by the manufacturer and were previously used in peer reviewed works.

Techniques: Expressing, Immunofluorescence, Transfection, Staining, Control, Strep-tag

Journal: Oncogene

Article Title: Chk1 is required to maintain claspin stability.

doi: 10.1038/sj.onc.1209447

Figure Lengend Snippet: Figure 1 Claspin is rapidly degraded in the cell. (a), Hela cells were transfected with S-FLAG-tagged Claspin. Cells were incu- bated with 100 mg/ml cycloheximide (CHX) for the indicated periods. Western blots were performed on whole cell lysates using anti-FLAG and anti-BACH1 antibodies. (b) HeLa cells were treated with 100 mg/ml cycloheximide for various lengths of time. Western blots were performed on whole cell lysates using anti- Claspin and anti-BACH1 antibodies. Relative Claspin protein levels were determined by densitometry and were normalized to that obtained in nontreated samples (0 h control). (c) HeLa cells were incubated with different doses of cycloheximide (CHX) for 5 h. Western blots were performed on whole cell lysates using anti- Claspin, anti-Chk1 and anti-Actin antibodies.

Article Snippet: Anti-Chk1 antibodies for Western blot were form Santa Cruz Biothecnology (Santa Cruz, CA, USA) and anti-phospho-Chk1 (Ser317) antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Transfection, Western Blot, Control, Incubation

Journal: Oncogene

Article Title: Chk1 is required to maintain claspin stability.

doi: 10.1038/sj.onc.1209447

Figure Lengend Snippet: Figure 2 Claspin is degraded by the proteasome-dependent pathway. (a) HeLa cells were treated with 10 or 25 mM of the proteasome inhibitor MG132 for 5 h. Whole cell lysates were subjected to Western blots with anti-Claspin, anti-BACH1, and anti-Actin antibodies. (b) HT1080 cells and HT1080 cells trans- fected with S-FLAG-tagged Claspin were treated for 5 h with 10 mM MG132. Whole cell lysates were subjected to Western blots with anti-S and anti-Claspin antibodies. (c) U20S cells were treated with 10 mM MG132 for 5 h. Whole cell lysates were subjected to Western blots with anti-Claspin and anti-Chk2 antibodies. (d) HeLa cells were treated with cycloheximide (CHX) for the indicated time points in the presence or absence of 10 mM MG132. MG132 was added 4 h before cells were lysed. Western blots were performed on whole cell lysates using anti-Claspin, anti-Chk1 and anti-RPA antibodies. Relative Claspin protein levels were determined by densitometry and were normalized to that obtained in nontreated samples (0 h control, no MG132). In the graph, samples with and without MG132 were normalized to their own control (0 h, 100%).

Article Snippet: Anti-Chk1 antibodies for Western blot were form Santa Cruz Biothecnology (Santa Cruz, CA, USA) and anti-phospho-Chk1 (Ser317) antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Western Blot, Control

Journal: Oncogene

Article Title: Chk1 is required to maintain claspin stability.

doi: 10.1038/sj.onc.1209447

Figure Lengend Snippet: Figure 3 Inhibition of Chk1 expression and activity decreases Claspin levels in the cell. (a) HeLa cells were treated with control, Claspin and Chk1 siRNA. Whole cell lysates were subjected to Western blots with anti-Claspin, anti-Chk1 and anti-Chk2 antibodies. (b) HeLa cells were treated with 300 nM UCN01 or 3 mM caffeine for 6 h. Western blots were performed in whole cell lysates using anti- Claspin and anti-Chk2 antibodies. (c) HeLa cells were treated with different doses of UCN01 for 5 h. Western blots were performed in whole cell lysates using anti-Claspin, anti-Chk1, and anti-Actin antibodies. Relative Claspin protein levels were determined by densitometry and were normalized to the Actin levels. (d) upper panel, HCC1937 cells and HCC1937 cells reconstituted with myc- tagged BRCA1 were treated with or without UCN01 for 16 h as indicated. Whole cell lysates were subjected to Western blottingh with anti-Claspin and anti-Chk1 antibodies. Lower panel, expression of BRCA1 in HCC1937 cells. BRCA1 was immunoprecipitated from HCC1937 and HCC1937 reconstituted with myc-epitope-tagged BRCA1 and blotted with either anti-BRCA1 or anti-myc antibodies. Western blot with anti-Chk2 antibody was included as a control. (e) HeLa cells were treated with hydroxyurea in the presence or absence of 300 nM UCN01. Claspin was immunoprecipitated and Western blots were performed using anti-Claspin antibody.

Article Snippet: Anti-Chk1 antibodies for Western blot were form Santa Cruz Biothecnology (Santa Cruz, CA, USA) and anti-phospho-Chk1 (Ser317) antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Inhibition, Expressing, Activity Assay, Control, Western Blot, Immunoprecipitation

Journal: Oncogene

Article Title: Chk1 is required to maintain claspin stability.

doi: 10.1038/sj.onc.1209447

Figure Lengend Snippet: Figure 4 Inhibition of Claspin expression by Chk1 siRNA and UCN01 is reversed by proteasome inhibition. (a) HeLa cells were treated with control and Chk1 siRNA. 67 h after the first siRNA transfection cells were treated with MG132 and 5 h later cells were collected and lysed. Whole cell lysates were subjected to Western blots with anti-Claspin, anti-Chk1 and anti-RPA antibodies. (b) HeLa cells were treated with 300 nM UCN01 for 3 or 6 h. MG132 was added for the last 4 h of UCN01 treatment. Western blots were performed in whole cell lysates using anti-Claspin and anti-Chk1 antibodies. (c) Cell cycle distributions after siRNA treatments (72 h) and after 6 h incubation with UCN01 were determined by FACS analysis and summarized below. Relative Claspin protein levels were determined by densitometry and were normalized to that obtained in non-treated samples (control) or samples transfected with control siRNA.

Article Snippet: Anti-Chk1 antibodies for Western blot were form Santa Cruz Biothecnology (Santa Cruz, CA, USA) and anti-phospho-Chk1 (Ser317) antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Inhibition, Expressing, Control, Transfection, Western Blot, Incubation

Journal: Oncogene

Article Title: Chk1 is required to maintain claspin stability.

doi: 10.1038/sj.onc.1209447

Figure Lengend Snippet: Figure 5 Overexpression of Chk1 increases Claspin stability. (a) HeLa cells were transfected with empty vector (C) or vectors encoding HA-tagged Chk1 or HA-tagged Chk2. Whole cell lysates were subjected to Western blots with anti-Claspin, anti-Chk1, and anti-HA antibodies. (b) Hela cells were transfected with vector encoding S-FLAG-tagged Claspin in combination with empty vector (C), or vectors encoding HA-tagged Chk1 or HA-tagged Chk2. Whole cell lysates were subjected to Western blots with anti- FLAG, anti-HA antibodies, and anti-RPA antibodies. (c) Cell cycle distributions in the presence of empty vector (control) and transfected HA-Chk1 and HA-Chk2 were determined by FACS analysis and summarized below. Relative Claspin protein levels were determined by densitometry and were normalized to that obtained in samples transfected with empty vector.

Article Snippet: Anti-Chk1 antibodies for Western blot were form Santa Cruz Biothecnology (Santa Cruz, CA, USA) and anti-phospho-Chk1 (Ser317) antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, Control

Journal: Oncogene

Article Title: Chk1 is required to maintain claspin stability.

doi: 10.1038/sj.onc.1209447

Figure Lengend Snippet: Figure 6 Claspin regulates late Chk1 activation following hydro- xyurea treatment (a) HeLa cells were transfected with control, Claspin and Rad17 siRNA. After 72 h, cells were treated for 1, 2 or 4 h with 10 mM hydroxyurea. Whole cell lysates were subjected to Western blots with antibodies against S317-Chk1, Claspin, Rad17 and Chk2. (b) HeLa cells were transfected with control and Claspin siRNA. After 72 h, cells were treated for 1, 2, 4 or 6 h with 1 mM hydroxyurea. Whole cell lysates were subjected to Western blots with antibodies against S317-Chk1 and Actin. (c) upper panel, U20S cells were transfected with control and Claspin siRNA. After 72 h, cells were treated for 1, 2, 4 or 6 h with 1 mM hydroxyurea. Whole cell lysates were subjected to Western blots with antibodies against S317-Chk1 and Actin. Relative phosphorylated Chk1 levels were determined by densitometry and were normalized according to Actin levels. Lower panel, expression of Claspin in U20S cells transfected with control and Claspin siRNA. Claspin was immunoprecipitated from U20S cells and Western blots were performed with anti-Claspin and anti-Actin antibodies.

Article Snippet: Anti-Chk1 antibodies for Western blot were form Santa Cruz Biothecnology (Santa Cruz, CA, USA) and anti-phospho-Chk1 (Ser317) antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Transfection, Control, Western Blot, Expressing, Immunoprecipitation

Journal: Biochemical Journal

Article Title: Ataxia telangiectasia mutated- and Rad3-related kinase drives both the early and the late DNA-damage response to the monofunctional antitumour alkylator S23906

doi: 10.1042/bj20101770

Figure Lengend Snippet: Figure 3 S23906 induces rapid activation of the ATR/Chk1 pathway in replicating cells

Article Snippet: The phospho-Thr68-Chk2 (#2661), Chk2 (clone 1C12, #3440), phospho-Ser317-Chk1 (#2344), phospho-Ser345-Chk1 (#2341), Chk1 (clone 2G1D5, #2360), phospho-Ser1981-ATM (clone 10H11.E12, #4526), ATM (clone D2E2, #2873), RPA32 (clone 4E4, #2208) and RPA70 (#2267) -directed antibodies were purchased from Cell Signaling Technology.

Techniques: Activation Assay

Journal: Molecular Biology of the Cell

Article Title: The nucleolus stress response is coupled to an ATR-Chk1–mediated G2 arrest

doi: 10.1091/mbc.E12-12-0881

Figure Lengend Snippet: The arrest is ATR-Chk1 pathway dependent. Cells were pretreated with either caffeine or UNC-01, subjected to nucleolar stress in the presence or absence of either inhibitor, and then cultured an additional 20 h in medium lacking actinomycin but with either inhibitor. (A) Images after a 4-h nucleolar stress. Left set of four, caffeine; right set of four, UCN-01. Scale bar, 100 μm. (B) Multicolor FACS analyses of the cells shown in A. (C) Cells were exposed to ATR-Chk1 inhibitors for 2 h and then actinomycin (with continued inhibitors) for 4 h and then examined at 0, 8, 16, or 24 h. Scale bar, 50 μm.

Article Snippet: The primary antibodies, dilutions, and suppliers were as follows: phospho–histone H2A.X (Ser-139; JBW301) mouse monoclonal antibody (1:200; Millipore, Billerica, MA) and phospho-Chk1 (Ser-317; D12H3) rabbit monoclonal antibody (1:200, Cell Signaling Technology, Beverly, MA).

Techniques: Cell Culture

Journal: Molecular Biology of the Cell

Article Title: The nucleolus stress response is coupled to an ATR-Chk1–mediated G2 arrest

doi: 10.1091/mbc.E12-12-0881

Figure Lengend Snippet: Elevation of Chk1 phosphorylation after 2–4 h of nucleolar stress. Cells were treated with either actinomycin D or doxorubicin for the various times indicated. Phospho-Chk1 was detected by immunofluorescence 20 h after treatment. Images were captured along with Fucci colors. Scale bar, 50 μm.

Article Snippet: The primary antibodies, dilutions, and suppliers were as follows: phospho–histone H2A.X (Ser-139; JBW301) mouse monoclonal antibody (1:200; Millipore, Billerica, MA) and phospho-Chk1 (Ser-317; D12H3) rabbit monoclonal antibody (1:200, Cell Signaling Technology, Beverly, MA).

Techniques: Phospho-proteomics, Immunofluorescence