Fig. 8

Co-expression of RhEcR and RhUSP induced cell death of HEK293T cells by apoptotic signals. a, b, c The results showed that different isoforms of RhEcR can interact with different RhUSP isoforms in HEK293T. a The expression of each isoform of Flag-RhEcR and HA-RhUSP in HEK293T cells. b Isoforms of Flag-RhEcR were detected by co-immunoprecipitation of HA-RhUSP. c Isoforms of HA-RhUSP were detected by co-immunoprecipitation of Flag-RhEcR. d The complex of GFP-RhEcR1 and mCherry-RhUSP1 induced cell death in HEK293T cells. GFP, mCherry, and GFP-RhEcR1 together with mCherry-RhUSP1 were transfected in HEK293T for 24 h. Images were obtained using a fluorescence microscope. e GFP-RhEcR1 and mCherry-RhUSP1 were co-located in the nucleus. GFP-RhEcR1 and mCherry-RhUSP1 were co-expressed in HEK293T for 24 h. Images were obtained using a fluorescence microscope. f, g The complex of Flag-RhEcR1 and HA-RhUSP1 induced cell death through the activation of the apoptosis pathway in HEK293T. Cleaved caspase-3 was detected during co-expression of Flag-RhEcR1 and HA-RhUSP1 and etoposide was induced. The agonist of apoptosis, etoposide, and co-expression of Flag-RhEcR1 and HA-RhUSP induced cell death. Co-expression of Flag-RhEcR1 and HA-RhUSP1 together with the caspase inhibitor, zVAD, inhibited cell death. In f, the data represent the mean ± SD of the experiments performed in triplicate one-way ANOVA multiple Comparisons were used to identify significant differences between groups (***P < 0.001) (ANOVA: Flag-EcR1/HA-USP1 vsFlag/HA, F (2, 6), P < 0.001; Flag-EcR1/HA-USP1 vs Flag-EcR1/HA-USP1/DMSO, F (2, 6), P > 0.9999; Flag-EcR1/HA-USP1/DMSO vs Flag-EcR1/HA-USP1/ZVAD, F (2, 6), P < 0.001; Blank vsEtoposide+, F (2, 6), P < 0.001)