Fig 2: A schematic diagram of perinatal to adult transition of OPCs. In developing rat optic nerves, thyroid hormone inhibits the proliferation of perinatal OPCs. In normoxia, cells differentiate into oligodendrocytes. On the other hand, in a hypoxic niche, quiescent OPCs sustain their potential capacities of proliferation and differentiation as adult somatic stem cells (Adult OPCs). Hypoxia-inducible factors-mediated Runx1 expression and resultant p15/INK4b induction contributes to development of adult OPCs.

Fig 3: Runx1 dictates the cell cycle deceleration of OPCs in hypoxia. (a) P7 rat OPCs were cultured without TH in 1.5% O2 conditions for 12 days, then the cells were co-transfected with anti-p15/INK4b siRNA (si-p15/INK4b) or siRNA against the gene of each transcription factor and pMaxGFP. Cells were cultured with TH in 1.5% O2 conditions for another 4 days. The number of GFP+ cells in each clone was counted. Data was normalized against the average number of negative control (cells transfected with non-target siRNA; si-NT). *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired Student’s t-test, n = 3). (b) To investigate the immediate early responses of the genes of the deceleration of cell cycle related transcription factors, P7 rat OPCs cultured without TH for 10 days in 1.5% O2 were pre-treated with cycloheximide (50 μg/ml) for 6 hours, after then TH was added to the medium. Cells were harvested at 0, 0.5, 1 and 2 hours later. RT-PCR assay was carried out. GAPDH was used as a negative control. (c) P7 rat OPCs were infected with retrovirus vector Klf9-IRES-ZsGreen and were cultured for 7 days without TH in 1.5% or 3% O2 conditions. Cont; empty vector RetroX-IRES-ZsGreen1 infected OPCs, Klf9; Klf9 over expressing cells. *P < 0.001 (unpaired Student’s t-test, n = 3). (d) P7 rat OPCs cultured with (+) or without TH (−) in 1.5% O2 for 1, 4 and 15 days, or in 3% O2 for 15 days. Then RT-PCR assay was carried out. GAPDH was used as an internal control. (e) Freshly prepared P7 OPCs were cultured without TH in 3% O2 conditions overnight. Then cells were infected with Runx1 (Runx1b-IRES-ZsGreen) over-expressing retrovirus vector (Runx1) or ZsGreen expressing empty vector (Control). Cells were cultured for 7 days with or without TH in 3% O2. The number of ZsGreen+ cells in each clone was counted. *P < 0.005, **P < 0.001 (unpaired Student’s t-test, n = 3). (f) OPCs overexpressing Runx1 (green) in (d) were stained with the nuclear stain DAPI (blue) and rabbit anti-p15/INK4b antibodies (red). Scale bar: 100 μm.