Fig. 4: DUB3 stabilizes YAP1 and promotes tumor progression.

A Control (Ctrl) or DUB3-depleted A2780 and SKOV3 cells were generated. Immunoblotting was performed to examine protein levels of YAP1 and DUB3. B mRNA levels of YAP1 target genes CTGF, CYR61, and ANKRD1 were measured in Control (Ctrl) or CK2α-depleted A2780 and SKOV3 cells by qRT-PCR (n = 3). C Protein levels of YAP1 and DUB3 were measured in control (Ctrl) and DUB3-depleted PANC-1, MDA-MB-231, H460, 786-O and LoVo cells by immunoblotting. D mRNA levels of YAP1 were measured in Control (Ctrl) or DUB3-depleted A2780 and SKOV3 cells by qRT-PCR (n = 3). E Control (Ctrl) or DUB3-depleted A2780 cells were subjected to vehicle or MG-132 (10 μM) for 10 h. YAP1 and DUB3 levels were measured by immunoblotting. F Control (Ctrl) or DUB3-depleted A2780 cells were treated with cycloheximide (200 μg/mL). Cell lysates were collected at the indicated times, and YAP1 protein levels were measured by immunoblotting (n = 3). The relative level of YAP1 to Actin was analyzed by image J. G Indicated constructs were transfected into cells, and His-tagged ubiquitin was pull-downed by Ni-NTA beads. The ubiquitination level of YAP1 was examined by immunoblotting. H The indicated constructs were transfected into cells, and MG-132 (10 μM) was administered for 10 h. Flag-YAP1 was immunoprecipitated using anti-Flag affinity gel and then was mixed with recombinant GST, GST-DUB3 or GST-DUB3 C89S. The polyubiquitination of YAP1 was examined by immunoblotting. A Coomassie Brilliant Blue (CBS) staining was performed to assess the levels of GST or GST-fusion proteins. I Vector (V) or Flag-YAP1 were transfected in Control (Ctrl) or DUB3-depleted A2780 cells, and the level of YAP1 was examined by immunoblotting. J The proliferation of cells generated in (I) was measured and analyzed. K Indicated concentrations of Cisplatin were treated into cells generated in (I), and a CCK8 assay was used to measure cell survival. L, M Cells in (I) (1 × 10⁶ per mice) were subcutaneously implanted into nude mice (n = 6). When tumors grew to around 150 mm³, mice were randomly grouped and administered with saline or cisplatin (5 mg/kg i.p. every 3 days). Tumor weights were measured (L) and analyzed (M).