Control sirna sictr

Control siRNA (siCtr) and siRNA targeting Nrf2 (siNrf2, cat# sc-156128) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). PC12 cells were transfected with siCtr or siNrf2 using the Lipofectamine 2000 transfection reagent and Opti-MEM I reduced serum medium (Thermo Scientific), as reported previously [42 (link),43 (link)]. Briefly, cells were incubated in media without antibiotic–antimycotic until reaching 70–90% confluency. Opti-MEM I and siRNA were mixed to make solution A, and Opti-MEM I and Lipofectamine 2000 were mixed to make solution B. The two solutions were then left in situ for 5 min and gently mixed to yield solution C. After 20 min, the culture media were replaced with fresh media without antibiotics containing solution C at a final concentration of 100 nM siRNA. After 6 h of transfection, the media containing siRNA were exchanged with fresh media containing antibiotic–antimycotic. After 36–48 h with daily media exchanges, the cells were used for further experiments.

Primary rat astrocytes were transiently transfected with RGC-32 siRNA (siRGC-32) or control siRNA (siCTR) (Santa Cruz Biotechnology) by using Lipofectamine 3000 (Invitrogen, Carlsbad, CA): 75 pmol of siRNA and 7.5 μl of Lipofectamine 3000 were diluted separately in Opti-MEM I Reduced Serum Medium (Gibco, Waltham, MA), incubated for 5 min at RT, and then combined and incubated for 10 min at RT. The mixture was then added to the astrocytes, which had been plated 1 day earlier on 6-well plates or 25-cm² flasks at a confluence of ~70% in antibiotic-free DMEM/Ham’s F-12 supplemented with 10% FBS, according to the manufacturer’s instructions. At 48 h after transfection, astrocytes were starved in serum-free DMEM/F-12 overnight and then treated with 10 ng/ml TGF-β or vehicle for the indicated periods of time.