Fig. 4
Supervillin-mediated HCC cell migration and invasion involves the RhoA/ROCK pathway during hypoxia. a. MHCC-97H cells were transfected with control or supervillin-specific siRNA for 48 h, exposed to hypoxia for 16 h, and lysates were assayed for the relative amounts of GTP-loaded (activated) Rac1, Cdc42, and RhoA. Cells that had been transfected with control RNAi were treated with a MEK inhibitor (PD0325901) or a p38 inhibitor (SB239063) before assaying for GTP-Rac1, Cdc42, and RhoA levels (right). b. The interaction between supervillin and RhoA. Cell lysates were prepared from HEK293 cells co-transfected with GFP-tagged supervillin and HA-tagged RhoA, as described in the Methods. Immunoprecipitation (IP) and immunoblotting (IB) were performed with anti-HA or anti-GFP antibodies. c. Mapping of the binding region of supervillin and RhoA. HEK293 cells were transfected with GFP-tagged supervillin and HA-tagged RhoA. Twenty-four hours after transfection, cells were harvested and lysed, and RhoA was immunoprecipitated with anti-HA. Pellets and lysates were immunoblotted with anti-HA or anti-GFP antibodies. d. The interaction between supervillin and RhoA. Total HEK293 cell lysates containing GFP-tagged SV1 (1–825) and HA-tagged RhoA(WT), RhoA(V14), or RhoA(N19) were prepared. IP and IB were performed with anti-HA or anti-GFP antibodies. e. MHCC-97H cells that had been transfected with control or supervillin-specific RNAi were treated with the ROCK inhibitor Y27632 2HCl, incubated for 18 h in 1% O2, and tested for migratory activity in Boyden Chamber Transwell assays. f. MHCC-97H cells that had been transfected with control or supervillin-specific siRNA were treated with the ROCK inhibitor Y27632 2HCl, exposed to hypoxia for 18 h, and tested for cell invasion through Matrigel in Boyden Chamber Transwell assays