Journal: The Journal of Biological Chemistry
Article Title: Targeting EphA2 under DNA damage causes mitotic bypass via p21 induction
doi: 10.1016/j.jbc.2026.111271
Figure Lengend Snippet: p21 is responsible for EphA2 knockdown–caused mitotic bypass. A and E , HeLa S3 cells were transfected with either siCtrl or siEphA2. After 12 h, the cells were treated with 0.5 μM ADR for 12 h, washed, and then further cultured for 48 h before Western blot analysis ( A ). Band intensities of cyclin B1 ( B ), p53 ( C ), and p21 ( D ) were quantified, and the ratios against the band intensity of α-tubulin are plotted. Three or four independent experimental results are shown as dots together with mean ± SD. p -values were determined using Welch's t test. E , HeLa S3 cells were transfected with either nontargeting or p21-targeting siRNA and further cultured for 48 h before Western blot analysis. At 24 h after transfection, the medium was changed with fresh medium. F and G , HeLa S3/tFucci(CA)5 cells were transfected with siEphA2, along with either siCtrl or sip21. After 12 h, the cells were treated with 0.5 μM ADR for 12 h, washed, and then further cultured for 24 h before time-lapse imaging for 24 h. G2 cells at the beginning of imaging were examined. In F , percentages of cells in G2 phase, cell death during G2 phase, and the cumulative percentage of mitotic bypass are shown as mean ± SD from three independent experiments. No cells entered M phase. G2, orange ; M/post-M, gray ; cell death, black ; mitotic bypass, blue . Percentages of mitotic bypass during imaging are shown in the graph ( G ) as mean ± SD calculated from three independent experimental results. p -values were determined using Student's t test. n = 40 cells per condition. ADR, adriamycin.
Article Snippet: Primary antibodies used for immunoblotting (IB), immunofluorescence (IF) or flow cytometry were as follows: mouse monoclonal anti-Chk1 (IB, 1:1000; G-4, sc-8408, Santa Cruz Biotechnology), anti-γ-tubulin (IF, 1:250; GTU-88, T6557, Merck), anti-p21 (IB, 1:1000, DCS60, 2946, Cell Signaling Technology), anti-p53 (IB, 1:1000; DO-1, sc-126, Santa Cruz Biotechnology), and anti-phospho-histone H3 (IF, 1:400; 6G3, 9706, Cell Signaling Technology) antibodies; rabbit monoclonal anti-EphA2 (IB, 1:1000; 6997S, Cell Signaling Technology), anti-phospho-Chk1 (Ser345, IB, 1:1000; 133D3, #2348, Cell Signaling Technology), and anti-phospho-EphA2 (Ser897, IB, 1:1000; D9A1, 6347, Cell Signaling Technology) antibodies; rabbit polyclonal anti-cyclin B1 (IB, 1:3000; IF and flow cytometry, 1:250; H-433, sc-752, Santa Cruz Biotechnology), anti-phospho-histone H2A.X (γH2AX, IB, 1:500; 2577S, Cell Signaling Technology), and anti-phospho KAP1 (Ser824, IB, 1:1000; A300–767A, Bethyl Laboratories, Montgomery) antibodies; and rat monoclonal anti-α-tubulin (IB, 1:4000; IF, 1:800; MCA78 G, Bio-Rad) antibody.
Techniques: Knockdown, Transfection, Cell Culture, Western Blot, Imaging
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