Fig. 6

Knocking down CRL4^CUL4A/DDB1 inhibits OCC growth by inducing mitophagy. a Cell proliferation determined by CCK8 assay in A2780CP cells after CRL4^CUL4A/DDB1 knockdown with or without shATG5. b Verification of ATG5 knockdown in A2780CP cells using shRNA. c, d Cell proliferation determined by CCK8 assay in A2780CP (c) and COC1/DDP (d) cells after CRL4^CUL4A/DDB1 knockdown with or without 1 mM of 3-MA treatment for 36 h. e Cell proliferation determined by colony formation assay in A2780CP cells (10,000 cells) after CRL4^CUL4A/DDB1 knockdown with or without 1 mM of 3-MA treatment for 36 h. f Cell proliferation determined by colony formation assay in A2780CP cells (3000 cells) after CRL4^CUL4A/DDB1 knockdown with or without 4 µM of CQ treatment for 24 h. g Cell proliferation determined by CCK8 assay in (left panel) A2780CP and (right panel) COC1/DDP cells after treatment with 0.5 µM MLN4924 for 24 h with or without 1 mM of 3-MA treatment for 36 h. h Cell proliferation determined by CCK8 assay in (left panel) A2780CP and (right panel) COC1/DDP cells after treatment with 0.5 µM MLN4924 for 24 h with or without 4 µM of CQ treatment for 24 h. i Cell proliferation determined by CCK8 assay in A2780CP cells after treatment with 0.5 µM MLN4924 for 24 h with or without 10 µM of Wortmannin treatment for 24 h. j Cell proliferation determined by CCK8 assay in A2780CP cells after ATG5 knockdown with or without 0.5 µM of MLN4924 treatment for 24 h. k Representative image of isolated A2780CP tumor xenografts from mice in cohorts. Female immunodeficient nude mice received a subcutaneous injection of 5 × 10⁶ A2780CP cells containing either shDDB1/shCUL4A or negative control constructs (NT). End-point tumors isolated after four weeks of growth are shown. l Quantification of (left panel) total tumor weight and (right panel) tumor volume for A2780CP xenografts (n = 5 per group). Statistical significance was determined using a t-test (*p < 0.05). m Immunohistochemical staining analysis of hematoxylin-eosin stain, DDB1, CUL4A, DRP1, and Parkin staining in NT or shCUL4A/DDB1 tumors. n Representative ventral bioluminescent images of nude mice injected with A2780CP-luc-puro cells transduced with indicated shRNA constructs on days 0, 7, 14, 21, and 28. o Longitudinal ventral bioluminescence measurement for all mice injected with A2780CP-luc-puro transduced with indicated shRNA. Data represent mean total counts (photons/sec) for each group ± SEM. (n = 6 mice per group). p Representative end point necropsy showing differential tumor size and distribution in mice injected with A2780CP cells. Scale bar, 1 cm. Visible tumors are circled in yellow. (For all panels in this figure, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, t-test)