Figure 1

ATM and p53 impede hepatic conversion. (A) 3TF-induced proliferation arrest in wild-type (WT) tail-tip fibroblasts (TTFs) was determined by BrdU incorporation and staining at day 3. GFP expression vector was used as a control. BrdU-positive cells were quantified, n = 6 fields. Scale bars, 100 μm. (B) 3TF-induced cell death was analyzed by Annexin V and propidium iodide (PI) staining at day 6. (C) Levels of phosphorylated p53 (p-p53, Ser15), p19 and p21 were characterized by western blot analyses. (D) mRNA levels of p53 target genes were quantified by qRT-PCR. (E) iHep cells were generated from WT, p53^−/−, p53^+/− and p19^−/− TTFs by 3TF transduction. iHep colonies were quantified 8 days after 3TF transduction, n = 4 independent experiments. (F) Elevated level of ATM phosphorylation at Ser1981 (p-ATM) was determined by immunofluorescence staining 48 h after 3TF transduction. The fluorescent intensity was quantified by LAS AF Lite software. Analyzed cell numbers: n = 32 for GFP group and n = 33 for 3TF group. Scale bars, 10 μm. (G-H) p-ATM (G) and p-p53 (H) levels were analyzed by the western blot assay in WT TTFs (G) or ATM^+/+ and ATM^+/− (H) TTFs 48 h after 3TF transduction. (I) Quantification of iHep colonies generated from ATM^+/+ and ATM^+/− TTFs. n = 3 for each group. Error bars indicate SD. ^*P< 0.05, Student's t-test.