Figure 2.

Downregulation of JDP2 suppressed the growth arrest induced by oxidative stress. (A) Schematic diagram showing details of the assay conditions. Wt MEFs cultured in low oxygen concentration (3% O₂) were infected with a lentivirus expressing shRNA for JDP2 (shJDP2) or the empty vector (plko). The infected cells were selected by puromycin in low oxygen concentration, then cultured in the environmental (20% O₂) or in the low oxygen concentration for 6 days. The cells were further cultured for 1 day in environmental oxygen concentration, and the proliferation rate was analyzed using an EdU incorporation assay. (B) Effect of shJDP2 on the proliferation activity of MEFs. The cells were cultured in the presence of EdU for 7 h. Whole cells and proliferating cells were visualized using Hoechst 33342 dye and a Click‐iT EdU Alexa Fluor Imaging Kit, respectively. We performed eight different experiments. Scale bar = 500 μm. (C) Effect of shJDP2 on cell growth of MEFs cultured for 1 and 7 days. The percentages of growing (EdU‐positive) cells against whole (Hoechst 33342‐stained) cells and the corresponding P‐values were calculated. We performed eight different experiments. The numbers of counted cells are shown in Table S1. (D) Expression of the mRNA for p16^Ink4a (left), Arf (middle), and JDP2 (right). Total RNA was extracted from the cells at the same time for the cell proliferation assay. The mRNA levels were analyzed by real‐time RT‐PCR using specific primers. Error bars in each graph represent the SD of the data.