Fig. S6

Nav3a depletion reduces cell migration in vitro. (A) Reduced Nav3a-protein and RNA levels in stable Nav3a-shRNA clone. Left: Pac-2 cells stably expressing either control shRNA or Nav3a shRNA were analyzed by immunofluorescence staining for Nav3a protein expression (left pictures and green in merge), actin (phalloidin staining in middle picture and red in merge) and with a nuclear stain (blue in merged picture). In stably expressing Nav3a shRNA cells, Nav3a protein is significantly reduced (white arrowheads). Middle panel: Nav3a protein expression in Pac-2 cells stable expressing control shRNA or Nav3a shRNA analyzed by Western blot. In Nav3a shRNA cells, Nav3a protein is significantly reduced. Right panel: Nav3a mRNA is reduced in Pac-2 cells stably expressing Nav3a shRNA compared with Pac-2 cells stably expressing control shRNA. Nav3a mRNA levels were quantified by real-time PCR and normalized to Elongation factor-1 (EF1) expression as a housekeeping gene. (B) Nav3a regulates migration of human hepatoma cells. Left: Human liver cells (HUH-7) were transfected with Nav3-siRNA and analyzed in an in vitro scratch-migration assay. Confluent monolayers were scratched and at indicated time points the migrated area was calculated. Nav3a inhibition significantly reduced the migratory capacity at 24 hours and 48 hours after transfection. Each graph represents an average of n=6-12 scratched areas, obtained from three separate experiments. Right: Nav3a mRNA is significantly reduced in HUH-7 cells transfected with Nav3a siRNA. Nav3a mRNA levels were quantified by real-time PCR and normalized to 18s RNA expression as a housekeeping gene. Data are expressed as mean ± s.e.m.