Loss of epithelial phenotype and development of dynamic structures of migration in HepG2 and Hep3B cells transfected with HNF1α siRNA. HepG2 and Hep3B cells were transfected with 10 nM of HNF1α siRNA (siH) or with a control siRNA (siC). A: Morphology of transfected HepG2 and Hep3B cells obtained by phase-contrast microscopy with a 10X objective after 72 h. Scale bar 100 μm. B: Actin stained using phalloidin (green) in HepG2 and Hep3B cells transfected with fluorescent control siRNA (red) or co-transfected with fluorescent control and HNF1α siRNA after 72 h. DNA is stained using DAPI (blue). Scale bar 10 μm. C: Time-lapse imaging showing dynamic cytoplasmic protrusions in HepG2 cells transfected with HNF1α siRNA compared to cells transfected with control siRNA, 72 h after transfection. Only 1 image every 50 min is shown here. Scale bar 20 μm. D, E: Expression of transcription factors involved in hepatocyte differentiation and of albumin, a marker of hepatocellular differentiation, in HepG2 (D) and Hep3B (E) cells transfected with HNF1α siRNA after 3 and 7 days for HepG2, compared with cells transfected with control siRNA. mRNA levels were analyzed by qRT-PCR and are expressed as n-fold difference in gene expression of HepG2 cells transfected with HNF1α siRNA (siH) relative to cells transfected with control siRNA (siC) (two-tailed t-test).