Fig. 1

OTUD6A upregulates CDC6 protein level. a, Quantitative analysis of CDC6 protein levels shown in Supplementary Fig. 1a. b, c, Increasing amounts of Flag-OTUD6A plasmids were transfected into HEK293 (293) cells, and the protein levels of endogenous CDC6 and exogenous OTUD6A were determined by Western blotting (b). The mRNA levels of CDC6 and OTUD6A were determined by qPCR (c), and the levels in empty Flag vector cells were set as 1. d, 293 and U2OS cells were transfected with the empty Flag vector or Flag-OTUD6A plasmid. An additional 24 h later, the cells were fixed and incubated with the Flag and CDC6 antibodies. Representative immunofluorescence images are shown (left). Scale bars, 20 μm. Quantification of the relative fluorescence intensity of CDC6 is shown (right), and the fluorescence intensity of CDC6 in Flag-OTUD6A untransfected or empty Flag vector-transfected cells was set as 1.e, f, CDC6 and OTUD6A expression levels were measured in the indicated 293 cells by Western blotting. g, Cytoplasmic, soluble nuclear and chromatin-bound nuclear fractions were extracted from the indicated cells using subcellular fractionation assay and detected by Western blotting. CF, cytoplasmic fractions; SNF, soluble nuclear fractions; CNF, chromatin-bound nuclear fractions. h, Chromatin-bound proteins (CBP) were extracted from the indicated 293 cells and analysed by Western blotting. i, Weight curves of WT and OTUD6A knockout (CKO) mice are shown (WT, n = 7; CKO, n = 7). j, Relative quantification of tissue weights from 8-week-old WT (n = 5) and CKO (n = 5) mice. k, l, The proliferation of the indicated mouse embryonic fibroblasts (MEFs) was measured by CCK8 assays (k) and EdU incorporation assays (l). m, Representative bright-field, H&E and immunofluorescence images of WT and CKO embryos at embryonic day (E) 13.5. The liver tissue is autofluorescent. Scale bar, 1 mm. n, CDC6 and OTUD6A expression levels were measured in WT and CKO mouse-derived MEFs by Western blotting. 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