Fig 1. Transfection of Hepa 1–6 cells with the growth-arrest specific 1 (Gas1) gene.

(A) Anti-HA immunofluorescence of Hepa 1–6 cells transfected with pcDNA3/CAG-HAGas1 in the absence of detergents to preserve the integrity of membranes. Nuclei were counterstained with DAPI. (B) Double immunofluorescent staining anti-HA/anti-BrdU of Hepa 1–6 cells transfected with pcDNA3/CAG-HAGas1. (C) Cell cycle analysis of GFP-positive HEPA 1–6 cells after transfection with pIRES/GFP empty vector. 10⁶ cells (areas indicated in the upper row) were sorted and subjected to cell cycle analysis after propidium iodide staining (lower row). (D) As (C), after transfection with pIRES/HAGas1. (E) Quantitation of the % of cells in the different stages of the cell cycle from the flow cytometry analysis. Experiments were done in triplicate. (F) Analysis of Ccne2 expression in Hepa 1–6 cells overexpressing Gas1. A representative RT-PCR showing Gas1 and Ccne2 expression in cells transfected with either empty pcDNA3/CAG (control) or pcDNA3/CAG-HAGas1 (HAGas1), 15 h after transfection. (G) qRT-PCR to determine CycE2 expression in cells as in (F). qRT-PCR was performed in triplicate from three independent experiments. Values were averaged and normalized to 18S rRNA. **, p<0.01. ***, p<0.001. In A and B the bar represents 11 μm.