Fig. 3

MORC2 overexpression promotes the sumoylation of C/EBPα and its subsequent degradation. a The sumoylation level of C/EBPα was increased by the ecotopic MORC2. HEK-293TT cells were co-transfected with His-C/EBPα, Flag-MORC2, and T7-SUMO1. Total lysates were subjected to western blot with indicated antibodies. b The deficient sumoylation mutant vector of His-C/EBPα-K161R was identified. HEK-293T cells were co-transfected with T7-SUMO1 and wild type of His-C/EBPα or SUMO deficient mutant of His-C/EBPα-K161R. Total lysates were subjected to western blot with indicated antibodies. c The overexpressed MORC2 increased the sumoylation level of C/EBPα, while didn’t affect the mutant C/EBPα-K161R level. HEK-293T cells were transfected with the expression vectors for Flag-MORC2, T7-SUMO1 and His-C/EBPα or His-C/EBPα-K161R, as indicated. Total lysates were subjected to western blot with indicated antibodies. d A cycloheximide (CHX) chase is carried out to really prove that MORC2 affects the stability of the wild-type and not K161R CEBP/α. We co-transfected wild-type His-C/EBPα or mutant C/EBPα-K161R and Flag-MORC2 into HEK-293T cells with the cycloheximide (CHX) treatment. After treatment with cycloheximide (CHX) (50 μg/ml) for 4 and 8 h, cell lysates were subjected to western blot with indicated antibodies. e The increased MORC2 affects wild-type not K161 mutant of C/EBPα stability. HEK-293T cells were transfected with the expression vectors for Flag-MORC2 and His-C/EBPα or His-C/EBPα-K161R, as indicated. After incubation with MG132 (10 μM) for 4 h, cell lysates were subjected to western blot with indicated antibodies. f MORC2 affects the sumoylated C/EBPα and its stability in HEK-293T cells. HEK-293T cells were transfected T7-SUMO1 and these vectors, as indicated in Fig. 3e, cell lysates were subjected to western blot with indicated antibodies. g MORC2 promotes the sumoylated C/EBPα levels and its stability in stable expressing SGC-7901 cell lines. These lentivirus-mediated cells were transfected with T7-SUMO1 and HA-ub, as indicated. After incubation with MG132 (10 μM) for 4 h, cell lysates were subjected to western blot with indicated antibodies. h, i IP assays demonstrate that MORC2 promotes the sumoylation of C/EBPα and its subsequent degradation in the presence of MG132. HEK-293T cells (h) and stable expressing SGC-7901 cell lines (i) were transfected with T7-SUMO1, as indicated. After incubation with MG132 (10 μM) for 4 h, cell lysates were subjected to IP assays with C/EBPα antibody, western blot detected with indicated antibodies. j The nuclear localization of C/EBPα was observed by confocal microscopy when MORC2 was overexpression. SGC-7901 cells were transiently co-transfected with His-C/EBPα and Flag-MORC2 for 36 h, as indicated, and incubated with Flag-tagged antibody to detect MORC2 expression, specific C/EBPα antibody to detect His-C/EBPα expression. Alexa Fluor 488 (green) and Alexa Fluor 546 (red) were used to detect His-C/EBPα and Flag-MORC2 (red) respectively. Yellow indicates co-localization. Blue, DNA dyed DAPI. h The nuclear localization of C/EBPα-K161R was observed by confocal microscopy when MORC2 was overexpression. SGC-7901 cells were transiently co-transfected with His-C/EBPα-K161R and Flag-MORC2 for 36 h, and incubated with Flag-tagged antibody to detect MORC2 expression, specific C/EBPα antibody to detect His-C/EBPα-K161R expression. Alexa Fluor 488 (green) and Alexa Fluor 546 (red) were used to detect His-C/EBPα-K161R and Flag-MORC2 (red) respectively. Yellow indicates co-localization. Blue, DNA dyed DAPI