Real-time monitoring of CYP3A4 expression in differentiated transgenic HepaRG cells according to EGFP fluorescence.

<p>(A) Strategy for hepatic differentiation of HepaRG cells <i>in vitro</i>. (B) Images of DsRed and EGFP in transgenic HepaRG clone 3 cells cultured in a 96-well plate at G14d, and after 5 days (D5d) and 10 days (D10d) of differentiation culturing. (C) Total fluorescence intensities of EGFP or DsRed in each of 6 wells during the culture of cells that have undergone 7 days of growing (G7d) until D12d. The different colored lines denote different wells. (D) qRT-PCR analyses showing the mRNA levels of EGFP, DsRed, endogenous CYP3A4, and endogenous CYP3A7 in HepaRG cells from G7d to D12d. The mean ± SD of three independent cultures were calculated, and the mRNA levels in cells relative to those in AL or FL (set at 100) are shown. (E) The morphology of EGFP-positive HepaRG cells at D14d is distinct from that of the other large flat cells. (F) Immunostaining of HepaRG C3 cells for anti-CYP3A4/7 antibody at D14d. (-) and (+) show cells before and after immunostaining, respectively. The scale bar is 100 µm in the image and 10 µm in the inset. Ph: phase-contrast images. (G) Western blot analysis of CYP3A4/7 and EGFP in HepaRG WT cells, HepaRG C3 cells, and HepG2 C87 cells. Signal intensities are shown relative to those in HepaRG C3 cells at D7d (set at 1).