Fig. 6

OTUD6A decreases sensitivity to chemotherapy via the CDC6-ATR-Chk1 pathway. a, The cell viability of the indicated T24 cells was determined after 48 h of continuous exposure to multiple concentrations of gemcitabine. The IC₅₀ value was defined as the concentration causing a 50% decrease in cell viability. The IC₅₀ values were estimated by nonlinear regression using a variable Hill slope model. b, The indicated T24 cells were treated with different concentrations of gemcitabine. Cell survival was determined by colony formation assays. c, Apoptosis was measured by TUNEL assays in the indicated T24 cells treated with or without 20 µg/L gemcitabine for 48 h. Representative images are shown (left). Scale bars, 50 μm. d, The amount of DNA strand breaks was quantified by alkaline comet assays in the indicated T24 cells treated with or without 20 µg/L gemcitabine for 48 h. Representative images are shown (left). Scale bars, 20 μm. e, The indicated T24 cells were treated with different concentrations of gemcitabine. Cleaved caspase-3 and γH2A.X protein levels were determined by Western blotting. f, The γH2A.X protein level was measured by immunofluorescence staining in the indicated T24 cells treated with or without 20 µg/L gemcitabine for 48 h. Representative immunofluorescence images are shown (left). Scale bars, 20 μm. g, The cell viability of the indicated T24 cells was determined after 48 h of continuous exposure to multiple concentrations of hydroxyurea (HU). h, The indicated T24 cells were treated with different concentrations of HU. Cell survival was determined by colony formation assays. i, Recovery of DNA replication activity was quantified by replication reinitiation assays. The indicated T24 cells were treated with 2 mM HU for 24 h and released into fresh medium for 4 h and 8 h. j, k, The effects of ATR and Chk1 inhibitors (VE-821 and GDC-0575) on regulating the sensitivity of OTUD6A-overexpressing UMUC3 cells to gemcitabine was determined by CCK8 (j) and TUNEL (k) assays. l, m, The indicated T24 cells were treated with different concentrations of gemcitabine (l) and HU (m). Cell survival was determined by colony formation assays. n, The amount of DNA strand breaks was quantified by alkaline comet assays in the indicated T24 cells treated with or without 20 µg/L gemcitabine for 48 h. o, Apoptosis was measured by TUNEL assays in the indicated T24 cells treated with or without 20 µg/L gemcitabine for 48 h. p, The γH2A.X protein level was measured by immunofluorescence staining in the indicated T24 cells treated with or without 20 µg/L gemcitabine for 48 h. q, Recovery of DNA replication activity was quantified by replication reinitiation assays. The indicated T24 cells were treated with 2 mM HU for 24 h and released into fresh medium for 4 h and 8 h. r, ATR-Chk1 pathway protein levels in the indicated T24 cells treated with 20 µg/L gemcitabine for 6 h were determined by Western blotting. s, Apoptosis was measured by TUNEL assays in the indicated T24 cells treated with or without 20 µg/L gemcitabine for 48 h. t, ATR-Chk1 pathway protein levels in the indicated T24 cells treated with 20 µg/L gemcitabine for 6 h were determined by Western blotting. u, Growth curves of the indicated subcutaneous T24 tumours treated with 50 mg/kg gemcitabine are shown. v, The indicated subcutaneous T24 tumours were weighed. All quantitative analyses were based on three independent experiments. The error bars indicate the SDs. *P < 0.05, **P < 0.01, ***P < 0.001, n.s. not significant, based on two-tailed Student’s t test