8 ohdg

A DNA dot-blotting was performed using extracted genomic DNA from a DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany). The DNA concentration were measured by a microplate reader (Epoch, BioteK, Winooski, VT, USA) at 260 nm/280 nm absorbance. Purified DNA were loaded onto 0.2 µM nitrocellulose membrane and hybridized for 2 h at 80 °C. The membrane was then blocked with 5% skim milk in 1X Tris-buffered saline (Bio-Rad) with 0.1% Tween 20 (TBST; Sigma-Aldrich) at room temperature for 1 h. After incubation with dsDNA (1:2000, Abcam) and 8-OHdG (1:200, Santa Cruz Biotechnology, Dallas, TX, USA) at 4 °C for overnight, membrane was washed in three changes of TBST for 15 min and then incubated with an anti-mouse or anti-rabbit horseradish-peroxidase-conjugated antibody (1:2500, Abcam) at room temperature for 2 h. Dot blots were visualized using electrochemiluminescence (Bio-Rad, Hercules, CA, USA) and evaluated with an Amersham Imager 600 (GE Healthcare Life Sciences, Uppsala, Sweden).

Cells were cultured in sterile Millicell EZ-Slide eight-well glass plates (EMD Millipore, #PEZGS0816). Before evaluation, the cells were washed with PBS, fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 30 min, blocked with 3% BSA for 1 h 30 min, and then incubated overnight at 4 °C with the primary antibodies. After washing, the cells were incubated for 1 h with secondary antibody. Images were acquired using a confocal fluorescence microscope (Zeiss, LSM 800). The following antibodies were used for immunofluorescence: ZO1 (Invitrogen #40–2200), MITF (Invitrogen, #PA5–38294), F-actin (Phalloidin FITC, Yepsen Biotechnology, #40735ES75), Metallothionein (Abcam, #ab12228), HMGB1 (Abcam, #ab18256), LC3B (CST, #3868), 8-OHdG (Santa Cruz, #SC-393871), Alexa Fluor 488-labeled donkey anti-rabbit IgG (CST, #4412), Alexa Fluor 488-labeled donkey anti-mouse IgG (CST, #4408), Alexa Fluor 594-labeled donkey anti-mouse IgG (CST, #8890), and Alexa Fluor 594-labeled donkey anti-rabbit IgG (CST, #8889).

After the behavioral observation, the mouse was deeply anesthetized, and the brain was removed and postfixed in 4% paraformaldehyde for 72 hours. Following the dehydration by using graded series of saccharose and embedding with OCT, coronal 16 μm thick sections (from -1.46 mm to -2.46 mm posterior to bregma) were made. Nonspecific binding was blocked by 10% normal goat serum. Sections were incubated with primary antibodies against SIRT1 (CST, USA), LC3B (CST, USA), TOM20 (CST, USA), 8-OHdG (Santa Cruz Biotechnology, USA), and VDAC (CST, USA) overnight at 4°C. Alexa Fluor 568 or 488 secondary antibodies (Invitrogen, USA) were further incubated for 2 hours at room temperature. The nucleus was identified with DAPI. Z-stack images were acquired by using a laser scanning confocal fluorescent microscope (20x, 40x oil, and 63x oil immersion objectives) (Carl Zeiss LSM 880, Germany) equipped with ZEN light software at 1,024 × 1,024 resolution. All quantitative analyses were performed from at least three independent experiments. Data were analyzed with ImageJ software.

The cell and tissue sections were washed with cold PBS and immediately fixed with cold methanol (−20°C) for 10 min, followed by incubation in 3% bovine serum albumin in PBS for 1 hr to block the nonspecific binding sites. A primary antibody to 8-OHdG (Santa Cruz) or γH2AX (Novus Biologicals) was added overnight at 4°C. After washing 3 times with 1X PBST, secondary antibodies and Alexa Fluor® 488-conjugated goat anti-mouse (1:1000, Life Technologies) were added for 1 hr at room temperature (Additional file 2: Table S3). Slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for analysis. For the DHE staining, the slides were incubated with the 5 μM DHE for 10 to 15 minutes in a dark chamber, on an orbital shaker at room temperature. After washing 3 times for 5 minutes/wash with 1X PBS and slight fixation for 4 to 8 minutes in 7% formaldehyde in 1X PBS, the slides were counterstained with DAPI, and images were captured using a fluorescence microscope.

RPMI-1640 media, mannitol, D-glucose and tangeretin were obtained from Sigma-Aldrich Chemical (St Louis, MO, USA), as were all other reagents, unless specifically stated elsewhere. Fetal bovine serum (FBS), trypsin-ethylenediaminetetraacetic acid and penicillin-streptomycin were purchased from Lonza (Walkersvillle, MD, USA). Rabbit polyclonal antibodies of FSP-1, HIF-1α and N-cadherin were obtained from Abcam Biochemicals (Cambridge, UK). Mouse monoclonal antibodies of α-SMA, E-cadherin, P-cadherin, ZO-1, nephrin, 8-OHdG), AQP1 and SOD2 were supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal podocin antibody and mouse monoclonal β-actin antibody were provided by Sigma-Aldrich Chemical. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG, goat anti-mouse and donkey anti-goat IgG were purchased from Jackson ImmumnoReserch Laboratories (West Grove, PA, USA). 4′,6-Diamidino-2-phenylindole (DAPI) was obtained from Santa Cruz Biotechnology.
tangeretin was dissolved in dimethyl sulfoxide (DMSO) for live culture with cells; a final culture concentration of DMSO was <0.5%.