Fig. 3: GASC1 positively regulates ROCK2 protein level through a mechanism dependent on its histone demethylase activity.
From: GASC1 promotes hepatocellular carcinoma progression by inhibiting the degradation of ROCK2
a High-density protein microarray screening analysis of cell lysates from cultured control and GASC1-depleted Hep3B cells. Proteins significantly downregulated by GASC1 depletion were summarized in the bar graph. b Immunoblotting (IB) analysis using the indicated antibodies in Hep3B GASC1 knockout (KO) cells. c, d IB analysis using the indicated antibodies in Hep3B (c), Huh7 CSC (d, left), and PLC/PRF/5 (d, right) CSC cells infected with shGASC1 and NT shRNA for 72 h. e, f qRT-PCR analysis of GASC1, ROCK2, and p65 mRNA levels in Hep3B GASC1 knockout cells (e) or HA-GASC1 over-expressed Huh7 and PLC/PRF/5 cells (f). Error bars represent SEM of three independent experiments. **P < 0.01 and ***P < 0.001 by Student’s t test. g–i IB analysis of whole-cell lysates (WCL) derived from Hep3B GASC1-KO cells (g), Huh7 and PLC/PRF/5 cells (h), and HEK293T cells (i) transfected with indicated plasmids for 48 h. j ROCK2 levels in HEK293T cells were monitored during the indicated time frame after transfection of HA-GASC1 plasmid (8 h to 120 h post transfection). k GASC1 and ROCK2 gene expression positively correlate with each other in HCC clinical specimens. l IB analysis of ROCK2 in Hep3B and PLC/PRF/5 CSCs after treatment with increasing doses of SD70, an inhibitor of the GASC1 histone demethylase activity, for 48 h (left panel); or 2.5 μM SD70 for increasing lengths of time as indicated (right panel).
