5 aza cdr

To determine the effect of 5-aza-CdR on HULP cells, cells were incubated in culture medium (day 0 = D₀) until achieving a confluence of 70% (day 3 = D₃). Then, they were starved in serum-free medium overnight and treated (day 4 = D₄) with different doses of 5-aza-dC (Abcam, Cambridge, UK) (n = 8): 0 μM (control), 2 μM (low-dose), 5 μM 5-aza-CdR (middle-dose), 10 μM (high-dose) for 72 h (day 7 = D₇). Medium was changed every 24 h.

Purified AT2 cells extracted immediately after birth (0 day) were plated out into 6-well plates and then cultured in environments of differing oxygen concentrations: a control (21 % O₂) or model (85 % O₂) group. Each group of cells was cultured in the presence of various concentrations of 5-Aza-2′-deoxycytidine (5-Aza-CdR, Abcam; 2.5, 5 or 10 μmol/L), Jumonji domain containing 3 (Jmjd3, Abcam; 10, 20 or 40 μg/L) or 3-Deazaneplanocin A (DZNep, Selleckchem, Shanghai, China; 1, 2.5 or 5 μmol/L) for 48 h, or treated with 5 μmol/L 5-Aza-CdR, 20 μg/L Jmjd3 or 1 μmol/L DZNep for 24, 48 or 72 h. Inhibitors and media were changed every 24 h until cells were collected. Cells of the negative control and model groups were cultured without inhibitors. After storage in liquid nitrogen, cells were transferred to a −80 °C freezer and then subsequently used for real-time PCR and Western blotting.