Figure 8

Knockdown of WNK1 in HUVEC cells significantly decreases the co-cultured hepatoma cell migration, and OSR1 overexpression can rescue the reduced cell migration caused by shWNK1 knockdown in endothelial cells. (a) The protein levels of WNK1 and OSR1 with WNK1 knockdown and/or OSR1 overexpression in HUVEC cells were detected by Western blot. (b) Quantification of WNK1 Western blot indicated that knockdown WNK1 (shWNK1-1 and shWNK1-2) in HUVEC cells significantly decreases the WNK1 protein levels. (c) Quantification of OSR1 Western blot indicated that overexpression of OSR1 in HUVEC cells significantly increases the OSR1 protein level, knockdown WNK1 (shWNK1-1 and shWNK1-2) in HUVEC cells significantly decreases the OSR1 protein levels. (d) HUVEC cells modulated the expression of WNK1 and OSR1 and were seeded in the lower chamber while HepG2 hepatoma cells were loaded in the upper chamber of the transwell plate. (e) Representative images of hepatoma cell migration. The scale bar is 200 µm in length. (f) Quantified data of the transwell migration assay shows that HUVEC cells with WNK1 knockdown significantly decrease the migrated hepatoma cells, indicating that WNK1 plays an important role in endothelial cells for stimulating hepatoma cell migration. Overexpression of OSR1 in shWNK1 knockdown HUVEC cells can revert the reduced hepatoma cell migration caused by shWNK1, indicating that OSR1 is downstream of endothelial WNK1 promoting hepatoma cell migration. We performed three replicates for each Western blot and transwell analysis. One-way ANOVA analysis followed by multiple analysis of variance was used to identify the statistical significance between groups. *: p ≦ 0.05; **: p ≦ 0.01; ***: p ≦ 0.001; ****: p ≦ 0.0001; ns: no-significance.