Coxii

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).

Rat joint tissue was fixed in 4% paraformaldehyde, decalcified in EDTA bone decalcifier and embedded in paraffin. The sections (7 μm) were stained with hematoxylin and eosin (H&E) and toluidine blue to detect proteoglycans. For immunohistochemistry, rat joint sections were blocked with 1% normal goat serum and stained with antibodies to IL-6 (1 μg/mL, Santa Cruz, CA, USA), COX-II (200 ng/mL, Santa Cruz, CA, USA) and isotype control antibody (1 μg/mL, Santa Cruz, CA, USA) at 4 °C overnight. After three washes in PBS, the secondary antibody (biotin-labeled goat anti-rabbit IgG) was applied for 1 h at room temperature. Staining was detected with 3, 3′-diaminobenzidine tetrahydrochloride and the sections were then counterstained with H&E and observed under a light microscope.