A549 and SPCA1 cells were purchased from the Chinese Academy of Sciences Shanghai Cell Bank and cultured in RPMI-1640 supplemented with 10% fetal bovine serum in a humidified atmosphere containing 5% CO2 at 37℃. The medium was changed at 2-day intervals and cells re-plated at 80–90% confluence. CIH or normoxia model in vitro was performed as our previous study [22]. Briefly, cells were cultured in a computer‐controlled incubator chamber attached to an external O2–CO2 computer‐driven servo controller (Biospherix, Lacona, NY), where the O2 concentration was altered between 0 and 21% every 30 min by injecting N2 or O2 with 5% CO2. The dissolved O2 inside the culture medium was monitored by a laser O2 probe (Biospherix) and the CIH reached 5% O2 and 21% O2 as hypoxic and normoxic values according to the sensing of the cells. Normal air conditions corresponded to 21% O2 and 5% CO2.Human Bach1 or control (Genechem, Shanghai, China) shRNA were transfected into A549 and SPC cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

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