Nrf 1

Cell extracts were prepared using lysis buffer¹³¹³. Immunoblotting was performed with antibodies against SDHA, NDUFA9 (Molecular Probes), NRF1, TFAM, VDAC1 (Santa Cruz Biotechnology) and α−tubulin (Sigma-Aldrich). Antibodies against ATF4, GRP78, total and phospho-eIF2α (Ser51) and CHOP/DDIT3 were purchased from Cell Signaling Technology. After incubation with the secondary antibodies, the membranes were washed 3 times, incubated with chemiluminescent HRP substrate Immobilon Western (Millipore) and scanned with a PXi gel imaging system.

Ovarian tissue samples were homogenized in PBS with a Teflon-glass homogenizer (Thomas Scientific, Swedesboro, NJ, USA) on ice and sonicated. Protein concentrations were determined by BCA protein assay (TIANGEN BIOTECH, Beijing, China). The protein samples were separated by SDS-PAGE and transferred onto nitrocellulose membranes (BioTrace™ NT, Port Washington, NY, USA). The membranes were blocked in 5% nonfat dry milk in tris-buffered-saline with tween 20 (TBST) for 1 h and incubated with a primary antibody against SIRT1, SIRT6, and FOXO3a (1:200 dilution, Santa Cruz Biotechnology, CA, USA), NRF1 (1:400 dilution, Santa Cruz Biotechnology, CA, USA), p53 (1:600 dilution, Santa Cruz Biotechnology, CA, USA) or β-action (1:1,000 dilution, Santa Cruz Biotechnology, CA, USA) over-night at 4°C, followed by the incubation with a secondary (horseradish peroxidase-conjugated) anti-rabbit or anti-mouse antibody (1:5,000 diluted) at room temperature for 1 h. Bands were visualized with a chemiluminescence reagent (Thermo Fisher Scientific, Waltham, MA, USA). Band intensities were analyzed using the Quantity One software (Bio-Rad Laboratories Pty. Ltd., Hercules, CA, USA). β-actin was used as a loading control.

24 hr after transfection of siRNA, cells were transfected with cDNA and cultured for a further 48 hr. 50 nM bortezomib was added 14 hr prior to cell lysis. Cells were lysed in buffer containing 42 mM Tris-HCl (pH 6.8), 1.72% SDS, 5.6% glycerol, 5% 2-mercaptoethanol, and 0.01% bromophenol blue (SDS sample buffer) for whole-cell lysate. The samples were subjected to SDS-PAGE, transferred to polyvinylidene fluoride membrane, and analyzed by immunoblotting. All images were taken using Fusion SL4 (M&S Instruments). Rabbit polyclonal antibody against DDI2 was raised by immunizing keyhole limpet hemocyanin (KLH) conjugated synthetic DDI2 C-terminal (residues 385–399) peptides. The following antibodies were purchased: Nrf1 (sc-13031; Santa Cruz), p97 (MA3-004; Invitrogen), GAPDH (sc-32233; Santa Cruz), Flag (F1804; Sigma Aldrich, St. Louis, MO).

One hundred thousand cells were plated in the 24-well plate with coverslip in 500 μL of medium, and after reaching 80% confluence, they were treated with different concentrations of Cr(VI) for 24 h. Cells were fixed in 4% paraformaldehyde, washed twice in PBS and then blocked for 30 min in blocking solution (10% goat serum, 1% BSA, 0.15% saponin in PBS) at room temperature. They were incubated with primary antibody PGC-1α (1:50, Santa Cruz Biotechnology, Santa Cruz, CA, USA), NRF-1 (1:50, Santa Cruz Biotechnology), COXII (1:50, Santa Cruz Biotechnology) and TFAM (1:50, Santa Cruz Biotechnology) for 24 h at 4 °C; washed 3 times with PBS, and incubated with Goat anti-Mouse IgG/IgM(H+L) secondary antibody, Alexa Fluor 488, 1:500, or Goat anti-Rabbit IgG/IgM(H+L) secondary antibody, Alexa Fluor 568, 1:500, (Thermo Fisher Scientific) for another 1 h at room temperature. Nuclei were stained with Hoechst for 5 min, and mounted on slides with coverslips. Images were taken using a Zeiss UV-LSM 510 confocal microscope (Zeiss, Oberkochen, Germany).

CP was purchased from Jiangsu Hengrui Medicine Co., Ltd. (Lianyungang, China). The ginseng sample (JSPACM-03-1) was collected from Jilin province and authenticated by Prof. Song-Lin Li via morphological and histological methods according to ginseng monograph in Chinese Pharmacopoeia (Version 2010). The voucher specimen was deposited in Department of Pharmaceutical Analysis and Metabolomics, Jiangsu Province Academy of Traditional Chinese Medicine. Reference compounds, including ginsenoside Re, Rg1, Rf, Ro, Rb1, Rc, Rb2, Rb3, Rd, 20(S)-Rg2, 20(S)-Rh2, 20(R)-Rg2 and 20(S)-Rg3, were purchased from Sichuan Victory Co. Ltd. (Chengdu, China). HPLC grade acetonitrile and methanol were purchased from Merck Company (Darmstadt, Germany). Antibodies for FXR, CYP7A1, CYP8B1, NTCP, OAT 2 and OAT 3, GST, BSEP, MRP 3, GCLM, GCLC, GS, NRF 1, NRF 2 and F4/80 were obtained from Santa Cruz Biotechnology (CA, USA). Brusatol was purchased from Beijing Zhili Biological Technology Co. Ltd. (Beijing, China). Ultra-pure water was prepared using Milli-Q SP system (Millipore, Bedford, MA, USA). All other reagents were of analytical grade and obtained from commercial sources.

Retinal slides were fixed with 4% paraformaldehyde at room temperature for 20 min and subsequently incubated with 0.5% Triton X-100 for 10 min. Then, the samples were treated with a blocking solution (5% normal goat serum and 2% bovine serum albumin in PBS) for 30 min to prevent nonspecific antibody–antigen binding. γ-H2AX, ligase IV, Nrf-1, CREB1 and P-CREB1 expression was detected using the antibody of γ-H2AX (Millipore), ligase IV (Santa Cruz), CREB1 (CST), P-CREB1 (CST) and Nrf-1 (Santa Cruz). For the fluorescence visualization of antibody reactions, the primary antibodies were detected using secondary antibodies labeled with the fluorochromes Alexa Fluor 555 or 488 (CST), while the nuclei were detected with DAPI.