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MLE-12 cells and exposure to hyperoxia

CS Chih-Hao Shen
JL Jr-Yu Lin
CL Cheng-Yo Lu
SY Sung-Sen Yang
CP Chung-Kan Peng
KH Kun-Lun Huang
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MLE-12 cells, the type II mouse-lung epithelial cell, were purchased from ATCC (Manassas, VA). Cells were cultured in a 50:50 mixed medium of DMEM and Ham’s F-12 supplemented with 4% FBS, insulin (5 μg/mL), transferrin (10 μg/mL), sodium selenite (30 nM), hydrocortisone (10 nM), β-estradiol (10 nM), HEPES (10 nM), and L-glutamine (2 mM). In transgenic studies, MLE-12 cells were cultured on 6-well plates. At 60–75% confluence, transient transfection was carried out using SPAK siRNA (50 nM) (Dharmacon RNA Technologies) as the SPAK-knockdown (SPAK-KD) or siCONTROL Non-Targeting siRNA (50 nM) as the negative control. In the hyperoxic group, cells were placed in an incubator filled with 95% O2 and 5% CO2 at 37 °C for 48 h. In the control group, cells were kept in 21% O2 and 5% CO2 at 37 °C for 48 h.

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