Fig. 2

Uev1 depletion inactivates AKT pathway signaling and reduces cell survival under serum starvation conditions in breast cancer cells. MDA-MB-231 or MCF7 cells were transfected with shRNA lentiviral particles against UEV1 (shUEV1) or non-specific target (shCK). shUEV1-1 and shUEV1-2 represent two independent stable shUEV1 MDA-MB-231 cell lines. shUEV1-8 represents a stable shUEV1 MCF7 cell line. a UEV1A transcript levels in shCK and shUEV1 lines were determined by qRT-PCR in MDA-MB-231 cells. b UEV1A transcript levels in shCK and shUEV1 line were determined by qRT-PCR in MCF7 cells. c AKT pathway proteins in whole-cell extracts (WCE) or nuclear fractions (N) were detected by western blot. The endogenous Uev1 was monitored by LN2B antibody. d Growth curve of non-specific target (shCK) and shUEV1 MDA-MB-231 cell lines (shUEV1-1 and shUEV1-2) under serum-deprived conditions. Then cells were harvested and cell numbers were determined as described in Fig. 1b, c. e Growth curve of non-specific target (shCK) and shUEV1 MCF7 cell line (shUEV1-8) under serum-deprived conditions. Then cells were harvested and cell numbers were determined as described in Fig. 1b, c. f Two shUEV1 MDA-MB-231 cell lines were transfected with the pcDNA4.0/TO/HA(+) vector expressing UEV1A (shUEV1-1 + UEV1A and shUEV1-2 + UEV1A). Non-specific shRNA targeted MDA-MB-231 cells were also transfected with the pcDNA4.0/TO/HA(+) vector to serve as a control. Cells cultured under serum-deprived conditions were harvested and cell numbers were determined as described in Fig. 1b, c. g The MCF7 shUEV1-8 cell line was transfected with pcDNA4.0/TO/HA(+) vector expressing UEV1A (shUEV1-8 + UEV1A). The non-specific shRNA targeted MCF7 cells were transfected with the vector alone (shCK + CK). Cells cultured under serum-deprived conditions were harvested and cell numbers were determined as described in Fig. 1b, c. Each sample was measured in triplicate and repeated at least 2 times. *P < 0.05; **P < 0.01