Fig 6. Human AECs change cell cycle proteins expression during hepatic differentiation.

Isolated amniotic cells were seeded in 6-wells plate and incubated with complete IMDM medium supplemented with 10% FBS (C) or hepatic differentiation medium (HD) for at least 30 days. At indicated times total RNA was extracted and mRNA expression of CYCLIN D1 (A), p53 (B) or p21 (C) were quantified by real time RT-PCR. CYCLOPHILIN and GAPDH were used as internal standards. (D) (E) At indicated times, cell extracts were prepared and proteins were separated on SDS-PAGE gels. Cyclin D1 and p53 expression were determined by Western-blot. Molecular weights were estimated using standard protein markers. Loading controls were performed by immunoblotting the same membranes with anti-GAPDH. Bands densitometry is shown in lower panels. Molecular weight (kDa) is indicated at the right of the blot. Representative results from three replicates are shown. (F) Amniotic cells were seeded in 24-wells plate and incubated in IMDM medium supplemented with 10% FBS (C) or hepatic differentiation medium (HD), during indicated times. At each time cells were fixed and p21 nuclear expression was detected using Alexa-488 conjugated secondary antibody. The nuclei were stained with DAPI. Percentage of positive cells is shown on the graph. Representative results from two replicates are shown. *p<0.05, **p<0.01, ***p<0.001 vs. control; #p<0.05, ##p<0.01, ###p<0.001 vs. respective control.