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. 2021 Oct 26;13(21):5373. doi: 10.3390/cancers13215373

Figure 5.

Figure 5

Depletion of TSPAN5 affects the actin/MRTF signaling axis. (A) Lysates from HepG2 DLC1 wt (DLC1 wt) and HepG2 DLC1 KO (DLC1 KO) cells transiently transfected with TSPAN5 siRNA (siTSPAN5) or control siRNA (sictrl) were immunoprecipitated with anti-active RhoA antibodies and immunoblotted with anti-RhoA antibodies. Lysates with an equal amount of total protein were directly immunoblotted with anti-RhoA antibodies. The knockdown efficiencies of DLC1 and TSPAN5 are shown in Figure 3B. (B) Immunoblotting with anti-actin antibodies and quantification of the ratio of F/G-actin in HuH7 cells transiently transfected with TSPAN5 siRNA (siTSPAN5) as compared to negative control siRNA (sictrl). Data are means ± SD (n = 3); ***, p < 0.001. (C) Immunofluorescence analysis and quantification of proximity ligation assay (PLA) for endogenous MRTF-A and FLNA in HuH7 cells transiently transfected with TSPAN5 siRNA (siTSPAN5) compared to negative control siRNA (sictrl). Scale bar, 10 μm. PLA signals were counted in 15 cells per condition. All data are means ± SD (n = 3); ***, p < 0.001. (D) MRTF target gene expression in HuH7 cells transfected with TSPAN5 siRNA (siTSPAN5) and scrambled siRNA (sictrl), determined by qRT-PCR using SM22-, GLIPR1- and TGFß1-specific primers and 18S rRNA primers for normalization. Values are means ± SD (n = 3); *, p < 0.05, **, p < 0.01 and ***, p < 0.001. (E) MRTF target gene expression in HuH7 cells treated as above and transfected with a GFP-empty vector (ctrl) or GFP-TSPAN5. Values are means ± SD (n = 3); *, p < 0.05, **, p < 0.01 and ***, p < 0.001. The whole Wester Blots are available at Figure S13.