Fig. 7

ML141 (ML) impact on hepatocyte differentiation is dependent of PI3K and Wnt5a signaling. hADSCs were induced to Hep-Dif for 28 days with or without ML141 (10 μM) for the indicated time of incubation (d-2/14 or d14/28). Cells were treated 24 h before adding ML141, and maintained with ML141, with: 1) inhibitors of PKA (H-89, 5μM), JNK (SP600125, 10μM), ERK (PD98059, 50μM), and PI3K (Wortmannin, 10μM); or 2) Wnt-antagonist Dkk1 (200 ng/ml, 24 h), Wnt3a (50 ng/ml, 24 h), and Wnt5a (100 ng/ml, 24 h). a mRNA expression of Wnt(s) and β-catenin expressed as fold variation relative to young at D0 after normalization to GAPDH. b Cell lysates (80–150 μg of protein) were separated by SDS-PAGE and immunoblotted with antibodies raised against phospho and total ERK/JNK/PKB/CREB. Protein expression profiling was determined during differentiation at D0/14/28 and results are expressed as fold variation of phospho/total levels relative to young at D0 after normalization to GAPDH. c Impact of H-89/SP/PD/WRT/Dkk1/Wnt3a/Wnt5a on the mRNA expression of the hepatic markers hepatocyte nuclear factor (HNF)4 and albumin (ALB) at D28 (hepatocyte-like cells; HLCs): results are expressed as fold variation relative to young untreated cells. Results are the mean ± SEM of three independent experiments performed in duplicate realized on 19 (young) and 20 (aged, aged+ML141) subjects. ^§*^#P < 0.05, ^§§**^## P < 0.01; ^§aged versus young, *aged treated with ML141 versus control, and ^#WRT or Wnt5a-treated cells versus control. WRT Wortmannin