anti phosphorylated chk1  (Cell Signaling Technology Inc)

Journal: Archives of biochemistry and biophysics

Article Title: Pericentriolar material 1 aggregation maintains cell survival upon prolonged replication stress.

doi: 10.1016/j.abb.2025.110383

Figure Lengend Snippet: Fig. 7. PCM1 promotes CHK1 activation to maintain cell survival upon prolonged replication stress. (A) Depletion of PCM1 did not affect CHK2 activation upon HU treatment. Extracts of control or PCM1-depleted cells were analyzed by western blotting with antibodies against phosphorylated CHK2, CHK2, and tubulin (loading control). (B) Inhibition of CHK2 did not affect U2-OS cell viability upon HU treatment. Quantitative results of relative cell numbers of HU-treated U2-OS cells in the presence of CHK2 inhibitor (CHK2i). (C) Depletion of PCM1 reduced CHK1 activation upon HU treatment. Extracts of control or PCM1-depleted cells were analyzed by western blotting with antibodies against phosphorylated CHK1, CHK1, and tubulin (loading control). (B) Inhibition of CHK1 reduced U2-OS cell viability upon HU treatment. Quantitative results of relative cell numbers of HU-treated U2-OS cells in the presence of CHK1 inhibitor (CHK1i). n.s.: no significance, ***: P < 0.001. Each experiment was repeated at least three times.

Article Snippet: The following antibodies were used for immunofluorescence and Western blot assays: anti-γ-tubulin (Sigma), anti-PCM1, anti-phosphorylated ATR, anti-ATR, anti-CHK1, anti-CHK2, anti-phosphorylated CHK1, anti-phosphorylated CHK2, anti-LC3 A/B (Cell Signaling), antip150glued, anti-GM130 (BD Biosciences), anti-Ku70, anti-α-tubulin, antiATM, anti-β-actin (GeneTex), anti-γ-H2AX, anti-p-ATM (Abcam), antiphosphorylated DNA-PKcs, anti-DNA-PKcs (Santa Cruz Biotech).

Techniques: Activation Assay, Control, Western Blot, Inhibition

Journal: Archives of biochemistry and biophysics

Article Title: Pericentriolar material 1 aggregation maintains cell survival upon prolonged replication stress.

doi: 10.1016/j.abb.2025.110383

Figure Lengend Snippet: Fig. 8. Autophagy maintains U2-OS cell viability upon prolonged replication stress. (A–B) Autophagy was induced by HU treatment in U2-OS cells. (A) LC3 puncta were examined by immunofluorescence staining with antibodies against LC3. DNA was stained with DAPI. Scale bar: 10 μm. (B) Quantitative results of (A). (C, upper panel) Extracts of HU-treated cells were analyzed by western blotting with antibodies against LC3 and actin. (C, lower panel) Quantitative results of upper panel. (D) Depletion of PCM1 alleviated HU-induced autophagy. Extracts of wild-type or PCM1-depleted (siPCM1) cells in the presence or absence of HU were analyzed by western blotting with antibodies against LC3 and actin. (E) Quantitative results of (D). (F) Inhibition of autophagy reduced U2-OS cell viability upon HU treatment. Quantitative results of relative cell numbers in wild-type or PCM1-depleted (siPCM1) cells in the presence or absence of HU. (G) Graphic abstract of this study. Prolonged replication stress promotes centrosome amplification independent of PCM1 aggregation. However, PCM1 promotes DNA damage signaling the ATM-CHK1 axis and autophagy to sustain cell survival. n.s.: no significance, ***: P < 0.001. Each experiment was repeated at least three times.

Article Snippet: The following antibodies were used for immunofluorescence and Western blot assays: anti-γ-tubulin (Sigma), anti-PCM1, anti-phosphorylated ATR, anti-ATR, anti-CHK1, anti-CHK2, anti-phosphorylated CHK1, anti-phosphorylated CHK2, anti-LC3 A/B (Cell Signaling), antip150glued, anti-GM130 (BD Biosciences), anti-Ku70, anti-α-tubulin, antiATM, anti-β-actin (GeneTex), anti-γ-H2AX, anti-p-ATM (Abcam), antiphosphorylated DNA-PKcs, anti-DNA-PKcs (Santa Cruz Biotech).

Techniques: Immunofluorescence, Staining, Western Blot, Inhibition, Amplification

Journal: International Journal of Molecular Sciences

Article Title: BCAT1 Associates with DNA Repair Proteins KU70 and KU80 and Contributes to Regulate DNA Repair in T-Cell Acute Lymphoblastic Leukemia (T-ALL)

doi: 10.3390/ijms252413571

Figure Lengend Snippet: BCAT1–depletion induces a dysfunctional DNA damage response following etoposide treatment. ( A ) Schematic representations of the plasmids encoding full-length (WT) and truncation mutants of XRCC6 (top). vWA: von Willebrand A domain; SAP: SAF-A/B, Acinus, and PIAS domain. HEK 293T cells stably expressing epitope-tagged BCAT1 were transfected with the indicated plasmid. Cell lysates were subjected to IP with anti-FLAG beads followed by immunoblot analysis with the indicated antibodies. The arrows indicate expected positions of the respective proteins, and asterisks (*) indicate non–specific bands. ( B ) Schematic representations of the plasmids encoding full-length (WT) and truncation mutants of BCAT1 (top). N: Branched–chain amino acid aminotransferase-like N-terminal domain; AT–IV: aminotransferase class IV domain; C: Branched-chain amino acid aminotransferase-like C–terminal domain. HEK 293T cells were transfected with HA–tagged XRCC6 and the indicated BCAT1 mutant plasmids. Cell lysates were subjected to IP with anti-HA beads followed by immunoblot analysis with the indicated antibodies. The arrows indicate expected positions of the respective proteins, and asterisks (*) indicate non-specific bands. ( C – E ) CCRF–CEM T-ALL cells transduced with shCTRL or sh BCAT1 were treated with 1 µM etoposide for the indicated time. Subsequently, whole cell lysates were collected and analyzed by immunoblotting for proteins implicated in ( C , D ) the activation of the DNA damage response (pDNA-PKcs, pATM, pCHK1, pCHK2, pTP53); ( E ) DNA damage (γH2AX) and apoptosis (cleaved PARP-1). Total DNA–PKcs and ATM are shown as loading controls ( C ). Total CHK2, total TP53, and GADPH are shown as loading controls ( D , E ). Phospho-protein/protein ratios are shown (top) in each panel. A graphical representation of the phospho-protein/protein ratios is also shown for selected proteins (right panels).

Article Snippet: Antibodies against tubulin (TU-02; sc-8035), c-myc (9E10; sc-40), and p53 (DO-1; sc-126) were from Santa Cruz Biotechnology (Dallas, TX, USA); antibodies recognizing FLAG epitope (#14793), BCAT1 (#88785), KU-80 (#2180), KU-70 (#4588), phosphorylated H2AX (pS139; #9718), phosphorylated DNA-PKcs (pS2056; #68716), total DNA-PKcs (#12311), phosphorylated ATM (pS1981; #5883), total ATM (#2873), phosphorylated CHK1 (pS345; #2348), phosphorylated CHK2 (pT68; #2197), total CHK2 (#6334), phosphorylated TP53 (pS15; #12571), and GADPH (#5174) were from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Western Blot, Mutagenesis, Transduction, Activation Assay

Journal: International Journal of Cancer

Article Title: Combined inhibition of RAD51 and CHK1 causes synergistic toxicity in cisplatin resistant cancer cells by triggering replication fork collapse

doi: 10.1002/ijc.35164

Figure Lengend Snippet: Synergistic toxicity evoked by combined treatment with the CHK1 inhibitor PF477736 (PF) and the RAD51 inhibitor B02 in J82 CisPt . (A) The viability of J82 CisPt cells was analyzed after treatment with differently combined low and moderate toxic doses of CHK1 i PF477736 and RAD51 i B02 as indicated. Cell viability was measured after a 72 h treatment period using the AlamarBlue Assay. Based on the data obtained from three independent experiments each performed in quadruplicate, the combination indices (CIs) were calculated using CompuSyn software (CI < 0.9 indicating synergistic effects, CI ≈1 additive effects and CI > 1.2 antagonistic effects). (B) Protein expression and activation of different apoptosis‐related factors were examined via Western Blot analyses using protein extracts of J82 CisPt cells treated for 6 or 24 h with 10 μM B02, 1 μM PF477736 or both substances. (C) 10 μM B02 and 1 μM PF477736 ± the pan caspase inhibitor QVD were added 24 h after seeding and after 24, 48 and 72h treatment period the induction of SubG1 fraction was analyzed by propidium iodide staining followed by detection by flow cytometry. Data presented are the mean + SD from three independent experiments. *** p ≤ .001; * p ≤ .05.

Article Snippet: The following antibodies were used: antibodies detecting Ser139 phosphorylated histone H2AX (γH2AX) (Millipore (Billerica, MA, USA) or Abcam [Cambridge, UK]), α‐tubulin, β‐actin (Santa Cruz Biotechnology [Santa Cruz, CA, USA]), Ser345 phosphorylated CHK1, CHK1, H2AX, cleaved caspase 7, caspase 7, PARP, Ser15 phosphorylated p53 (Cell Signaling [Danvers, MA, USA]), Ser4/8 phosphorylated RPA32, Ser824 phosphorylated KAP1 (Bethyl Laboratories [Montgomery, AL, USA]), Ser33 phosphorylated RPA32, RAD51, Pericentrin (Abcam [Cambridge, UK]), RPA32 (Millipore [Billerica, MA, USA]), rat anti‐BrdU (Abcam [Cambridge, UK]), mouse anti‐BrdU (BD [Franklin Lakes, NJ, USA]) and Ser10 phosphorylated histone 3 (pH 3) (Thermo Fisher [Waltham, MA, USA]).

Techniques: Alamar Blue Assay, Software, Expressing, Activation Assay, Western Blot, Staining, Flow Cytometry

Journal: International Journal of Cancer

Article Title: Combined inhibition of RAD51 and CHK1 causes synergistic toxicity in cisplatin resistant cancer cells by triggering replication fork collapse

doi: 10.1002/ijc.35164

Figure Lengend Snippet: Combining B02 or PF477736 with other CHK1‐ or RAD51‐inhibitors, respectively, likewise induces S‐phase arrest, replication stress, and DNA damage in J82 CisPt . J82 CisPt cells were co‐treated with 10 μM B02 + 1 μM LY2603618 (LY) (A) or 1 μM PF477736 (PF) + 30 μM RI(dl)2 (RI2) (B). Following 24 h treatment, propidium iodide‐based cell cycle analysis was performed by flow cytometry with emphasis on the proportion of cells in S‐phase. A total of 10,000 counts were measured for quantification. Induction of γH2AX and pRPA32 (S4, S8) was examined via Western Blot analyses with protein extracts of J82 CisPt cells treated for 6 or 24 h with the corresponding combination or mono‐treatments.

Article Snippet: The following antibodies were used: antibodies detecting Ser139 phosphorylated histone H2AX (γH2AX) (Millipore (Billerica, MA, USA) or Abcam [Cambridge, UK]), α‐tubulin, β‐actin (Santa Cruz Biotechnology [Santa Cruz, CA, USA]), Ser345 phosphorylated CHK1, CHK1, H2AX, cleaved caspase 7, caspase 7, PARP, Ser15 phosphorylated p53 (Cell Signaling [Danvers, MA, USA]), Ser4/8 phosphorylated RPA32, Ser824 phosphorylated KAP1 (Bethyl Laboratories [Montgomery, AL, USA]), Ser33 phosphorylated RPA32, RAD51, Pericentrin (Abcam [Cambridge, UK]), RPA32 (Millipore [Billerica, MA, USA]), rat anti‐BrdU (Abcam [Cambridge, UK]), mouse anti‐BrdU (BD [Franklin Lakes, NJ, USA]) and Ser10 phosphorylated histone 3 (pH 3) (Thermo Fisher [Waltham, MA, USA]).

Techniques: Cell Cycle Assay, Flow Cytometry, Western Blot