Tetraspanin TM4SF5 mediates loss of contact inhibition through epithelial-mesenchymal transition in human hepatocarcinoma
J. Clin. Invest. Sin-Ae Lee, et al. 118:1354 doi:10.1172/JCI33768 [Go to this article.]

Figure 2
TM4SF5-mediated morphological elongation involves RhoA inactivation. Whole-cell lysates were immunoblotted (A) or used for RhoA assay and immunoblots (C). Parental, SNU449Cp, or TM4SF5-expressing cells transfected with shRNA against GFP, scrambled sequence (Scr), or TM4SF5 (shTM4SF5) were harvested for immunoblots (B) or RhoA activity assay and immunoblots (D). (E) SNU449Tp cells were cotransfected with pEGFP and shTM4SF5. After 24 hours, cells were replated on 10% FBS/RPMI-1640–precoated coverslips. After an additional 24 hours, cells were stained for actin and DNA and analyzed by confocal microscopy. Scale bars: 20 μm. (F) Scheme showing the linkage from FAK to RhoA through RhoGAPs. FERM, FAK N-terminal band 4.1, ezrin, radixin, moesin homology domain; pxxp, proline-rich domain. (G) Increased association of RhoGAPs with FAK in SNU449Tp cells. Lysates from control (Cp) or SNU449Tp (Tp) cells were immunoprecipitated with anti-FAK antibody before immunoblot analysis. hc, heavy chain. (H) SNU449Tp cells were transiently transfected with (HA)₃-FAK WT or Y577F mutant. After 48 hours, lysates were prepared and immunoprecipitated with anti-HA antibody, before immunoblot analysis. (I) SNU449Cp cells were transfected with pEGFP-FLAG-GRAF or p190RhoGAP. After 24 hours, cells were replated on coverslips, as above. After an additional 24 hours, cells were double-stained for actin and then for FLAG-tag or GFP. Scale bars: 10 μm. (J) Cell extracts from Huh7 cells stably transfected with scrambled shRNA or shTM4SF5 were used in immunoblots. Data shown represent 3 different experiments.