p chk1  (Cell Signaling Technology Inc)

Journal: International Journal of Oncology

Article Title: Overcoming acquired doxorubicin resistance of ovarian carcinoma cells by verapamil-mediated promotion of DNA damage-driven cytotoxicity

doi: 10.3892/ijo.2026.5861

Figure Lengend Snippet: Effect of Topo II inhibitors on DNA damage formation and activation of DDR-related mechanisms in A2780 and A2780ADR cells. (A) At 24 h after treatment of logarithmically growing cells with the indicated concentrations of Doxo or Eto, the number of nuclear γH2AX-foci, 53BP1-foci, γH2AX/53BP1 co-localized foci and γH2AX pan-stained cells was analyzed. The upper part of the figure shows representative images (total magnification, ×1,000). Quantitative data depicted in the histogram are the mean ± SD from n=3 independent experiments with each five images being analyzed per experimental condition. * P≤0.05; ** P≤0.01; *** P≤0.001 (A2780 vs. A2780ADR); # P≤0.05; ## P≤0.01; ### P≤0.001 (treated vs. untreated control). Control experiments performed by use of 1st or 2nd antibody only or no antibody at all did not interfere with the signal of main interest (that is, nuclear foci; data not shown). (B) Logarithmically growing cells were treated with the indicated concentrations of Doxo for 24 or 72 h. Afterwards, the protein expression of DDR-related factors was analyzed by western blotting using EKR2 protein expression as loading control. Doxo, doxorubicin; Eto, etoposide; p-, phosphorylated; Chk1/2, checkpoint kinase 1/2; ERK2, extracellular regulated kinase; γH2AX, Ser139 phosphorylated histone H2AX; 2; Kap1, KRAB-associated protein 1; PARP, poly (ADP-ribose) polymerase; p21, cyclin-dependent kinase inhibitor 1; p53, tumor suppressor p53.

Article Snippet: The following primary antibodies were used: Copper transporting ATPase (ATP7A), extracellular regulated kinase 2 (ERK2), phosphorylated (p)-histone H3 (Ser10) from Thermo Fisher Scientific Inc., cleaved caspase-7 (Asp198), p-Chk1 (Ser 345), cyclin B1, galactosidase β (E2U2I), GAPDH (14C10), MDR1/ABCB1 (D3H1Q), p-P53 (S15), PARP, TopBP1(D8G4L), topoisomerase IIa (D10G9), 53BP1 and Ki67 were from Cell Signaling Technology Inc., pChk2 (T68) [Y171], copper uptake protein 1 (CTR1/SLC31A1) [EPR7936] and Rad51 from Abcam, γH2AX (Ser 139) clone JBW301, p-KAP-1 (S824) and p-RPA32 (S4/S8) from Bethyl Laboratories Inc., organic cation transporter-2 (OCT2) from Biozol Diagnostics Vertrieb GmbH, p16 (F-12) and p21 (C-19) from Santa Cruz Biotechnology, Inc. As secondary antibodies, horseradish peroxidase-conjugated secondary antibodies goat anti-mouse IgG and mouse anti-rabbit IgG were used (Rockland Immunochemicals Inc.).

Techniques: Activation Assay, Staining, Control, Expressing, Western Blot

Journal: Nature Communications

Article Title: Targeting de novo pyrimidine synthesis confers vulnerability to copper-mediated ATR inactivation in PARP inhibitor-resistant ovarian cancer

doi: 10.1038/s41467-026-70001-5

Figure Lengend Snippet: a Cell viability analysis of ES-2 parental and drug-adaptive cells treated with elesclomol-copper or olaparib at the indicated concentrations for 48 h. Drug-adaptive models were established by treating ES-2 cells with elesclomol-copper (200 nM) and olaparib (100 μM) individually or in combination for 7 days. Data are presented as the mean ± s.d.; two-way ANOVA; n = 5 technical replicates. Representative results from three biological replicates are shown. b Proliferation analysis of parental ES-2 cells and 7-day induced surviving cells. Data are presented as the mean ± s.d.; n = 3 technical replicates. Representative results from three biological replicates are shown. c Quantification of cell-cycle distribution in parental ES-2 cells and 7-day induced surviving cells. Data are presented as the mean ± s.d.; n = 3 technical replicates. Representative results from three biological replicates are shown. d GSEA of RNA-seq data comparing ES-2 parental and drug-adaptive cells. Statistical significance was assessed using GSEA with 1000 gene set permutations, without adjustment for multiple comparisons. e Western blot analysis of ATR-CHK1 and ATM-CHK2 pathways in ES-2 parental and drug-adaptive cells. Cells were pretreated with olaparib (10 μM) for 24 h before protein collection. Representative results from three biological replicates are shown. f Quantification of IF staining for p-ATR (S428) and p-CHK1 (S345). Cells were pretreated with olaparib (10 μM) for 24 h before IF assays. Data are presented as the mean ± s.e.m.; two-sided unpaired Student’s t -test; n = 5 biological replicates. g , h Bliss synergy score analysis for combinations of elesclomol-copper plus olaparib and AZD6738 plus olaparib in panels of parental and drug-adaptive ovarian cancer cell lines. Cell viability was measured after 48 h of combined drug treatment. Detailed treatment concentrations are provided in Supplementary Fig. . Violin plots display the distribution and median of all data points. For ( a ) and ( f ), exact p -values for all comparisons are shown directly in the figure. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were used for IF analysis: anti-p-ATR (S428) (1:100; AP0676, ABclonal), anti-ATR (1:200; ab313435, Abcam), anti-p-CHK1 (S345) (1:50; 2348, Cell Signaling Technology), anti-CHK1 (1:200; ab40866, Abcam), anti-γ-H2AX (1:50; AP0687, ABclonal).

Techniques: RNA Sequencing, Western Blot, Staining