Fig. 3

Puromycin-based selection of ProliHH. A Scheme of culture protocol. Puromycin was added at each passage for selection. B Phase-contrast photomicrographs of ProliHH with exposure to puromycin for 3 days. Different concentrations of puromycin (0, 1, 2, 10, 50 and 100 µg/mL) were added at 80% confluence. C Phase-contrast photomicrographs of ProliHH before and after 2 μg/mL puromycin. Puromycin was added when the cells reached confluence (Day 0) and removed 3 days after the addition (Day 3). D Growth curves of puromycin-treated ProliHH in duplicated experiments. Proliferative capacity was analyzed at each passage. Cells were passaged in the ratio of 1:4 at each passage (n = 2). "Population doubling" indicates the cumulative number of divisions of the cell population. E Phase-contrast photomicrographs of puromycin-treated ProliHH at passage 1, 5, 7, 13, 21 and 25. F Microscopic view of puromycin-treated ProliHH at passage 5 and 13. HE stain.G Immunocytochemical analysis of puromycin-treated ProliHH with an antibody to Ki67 (cell proliferation marker). H A senescence-associated beta-galactosidase stain of puromycin-treated ProliHH at passages 5 and 25. The number of β-galactosidase-positive senescent cells increased at passage 25. I Karyotypes of puromycin-treated ProliHH at Passage 24. Details are given in Supplemental Figure S3B