Figure 1.

Effects of culturing HepG2 and HepaRG in the absence or presence of collagen I for 32 and 48 h. A total of 32 h of culture is composed of 24 h attachment and 8 h of 2.5% BSA. A total of 48 h of culture is composed of 24 h attachment and 24 h of 2.5% BSA. Cell proliferation in (A) HepG2 and (B) differentiated and proliferating HepaRG. (C) Cell adhesion and proliferation in HepG2 were measured using the xCELLigence real-time cell analysis system for a period of five days. The plots shown are representative of at least three replicate experiments. (D) Mitochondrial respiration in HepG2 after 48 h of seeding was measured using a Seahorse XFe96 real-time extracellular flux analyzer. (E) Maximal respiration [1] in HepG2 was calculated from the oxygen consumption rate measured using a Seahorse XFe96 real-time extracellular flux analyzer. (F) Relative gene expression of integrin receptor subunits (ITGA2 and ITGB1). Protein expression of (G) the integrin receptor subunit (ITGA2, 48 h) and (H) phosphorylated focal adhesion kinase (P-FAK) were quantified using enzyme-linked immunosorbent assay. (I) Images showing cell morphologies of HepG2 and HepaRG in the absence or presence of collagen I were captured using a light microscope (objective magnification, x10). Data are expressed as mean ± SD. Statistical analyses were carried out using two-way ANOVA followed by Tukey’s post-hoc test. * p < 0.05, *** p < 0.001. ns, not significant. (A,B) n = 8, (D,E) n = 16, and (F–H) n = 3. O.D: optical density, Oligo: oligomycin, FCCP: carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, Rot: rotenone, A: antimycin A. BSA: bovine serum albumin. −Collagen I: absence and +Collagen I: presence of collagen I.