Nrf2 specific sirna

The selective antibiotics, zeocin was purchased from Invitrogen (CA, USA). 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and MitoTracker were purchased from Molecular Probes (Invitrogen, CA, USA). Antibodies against PGC-1α, Keap1, and Nrf-2, were purchased from Santa Cruz (Dallas, Texas, USA). Antibody against Hrd1 was purchased from Novus Biologicals (Littleton, CO, USA). Antibodies against caspase 3, cleaved caspase 3, Bax, Bcl2, phosphor-p53, total-p53, phosphor-GSK3β, totoal-GSK3β, phosphor-p38, phosphor-ERK1/2, phosphor-JNK, total-p38, total-ERK1/2, total-JNK, HO-1 and c-myc were all purchased from Cell Signaling Technology (Danvers, MA, USA). SB203580 and PD98059 were purchased from Calbiochem (Cat#559398 for SB203580 and Cat#513001 for PD98059, Darmstadt, Germany). N-acetyl-L-cysteine (NAC, Cat#A7250) and Thiazolyl Blue Tetrazolium (Cat#M2128) for MTT assay was purchased from sigma-aldrich. For over-expression of human PGC-1α in HK-2 cells, human PGC-1α/pCDNA4 plasmid was purchased from Addgene (Cat#10974, Cambridge, USA). Nrf-2 specific siRNA (Cat# sc-37030) and control siRNA (Cat# sc-37007) were purchased from purchased from Santa Cruz (Dallas, Texas, USA). DhamaFECT 1 Transfection reagent was purchase from GE Healthcare (Cat# T-2001-02, USA).

The cells were plated in 6-well plates at a density of 2 × 10⁵ cells/well in 2 mL DMEM. Next day, cells were transfected with Nrf2 specific siRNA (sense, 5′ GCACGGU GGAGUUCAUGATT 3′ and antisense, 5′ UCAUUGAACUCCACCGUGCCT 3′) (Santa Cruz Biotechnology, Inc., San Francisco, CA, USA). According to Lipofectamine^® 3000 manufacturer’s instructions (Invitrogen Life Technologies, Carlsbad, CA, USA), the cells were transfected with siRNA when they reached 50%–60% confluence in the plate. The cells were re-incubated in medium with 10% FBS after transfected 24 h. The knockdown effect of Nrf2 in these cells was verified by Western blot after transfected 24 h.