Figure 5

RNA interference, ELISA and ChIP experiments. (A): Relative expression of Smo, Gli1, Gli2 and Gli3 determined by qRT-PCR analyses in cultured hepatocytes of male C57BL/6-N mice in response to transfection of Smo siRNA (black bars) (n = 8) compared to nonsense transfection (white bars) (n = 8) after 48 h of incubation. Values are presented as relative means ± SEM; *, p < 0.05. (B): Relative expression of Ptch1, Gli3, Igf1 and Igfbp1 determined by qRT-PCR analyses in cultured hepatocytes of male C57BL/6-N mice in response to transfection with Ptch1 siRNA (black bars) compared to nonsense transfection (white bars) after 48 h (n = 10-12) and 72 h (n = 5-6) of incubation. Values are presented as relative means ± SEM; *, p < 0.05; **, p < 0.01; ***, p < 0.001. (C): Relative expression of Gli3 determined by qRT-PCR analyses in cultured hepatocytes of male C57BL/6-N mice in response to transfection of Gli3 siRNA (black bars) (n = 11) compared to nonsense transfection (white bars) (n = 11) after 72 h of incubation and relative expression of Igf1 and Igfbp1 in C57BL/6 N-hepatocytes after 72 h of incubation with Gli3 siRNA (black bars) (n = 8) compared to nonsense transfection (white bars) (n = 8). Values are presented as relative means ± SEM; *, p < 0.05; **, p < 0.01. (D): Supernatant analyses of secreted IGF-I and IGFBP-1 in cultured hepatocytes of male C57BL/6-N mice in response to transfection of Gli3 siRNA (black bars) (n = 6) compared to nonsense transfection (white bars) (n = 6) after 72 h of incubation. Values are presented as the means ± SEM; *, p < 0.05. (E): GLI3 binding site at -405 bp of the Igf1 promoter according to MotifMap. (F): qRT-PCR analyses of precipitated DNA. DNA immunoprecipitated with GLI3 Antibody (black), Histone H3 Antibody as positive control (grey), and IgG as a negative control (white) calculated relative to input.