The influence of quinoxalone on B16 cells observed by inverted microscope
B16 cells were plated onto glass cover slips in 6-well plates and treated with quinoxalone for 48 h. Then, cells were washed twice with PBS, fixed with 1% glutaraldehyde, stained with Hoechst 33342 for 15 min at room temperature. Nuclear morphology was examined by fluorescence microscope.
Fluorescence micrographs of B16 cells
B16 cells were treated without (A) and with quinoxalone (B: 5 μg/mL, C: 10 μg/mL) for 48 h. White arrow were the normal cells in A. White arrow were the apoptosis cells in B and C .
Effect of quinoxalone on DNA of B16 cells
B16 cells were treated with different dose of quinoxalone for 48h. Isolated DNA was analysed in agarose gel electrophoresis as described in Material and Methods. 200 bp DNA ladder marker (novoprotein, China) was used as marker (M) of DNA fragment size.
Effect of quinoxalone on mitochondrial transmembrane potential in B16 cells
After treatment without (A,control) and with 2.5 µg/mL (B) , 5 µg/mL(C), 10 µg/mL (D) quinoxalone for 48 h, the cells were double-stained with Rhodamine-123 and PI for 30 min, respectively. The percentages of PI negative and low-staining (Rh123-PI-) group represent the apoptotic cell group.
Effect of quinoxalone on the expression of Bax, Bcl-2 and P53 protein
Cells were treated without (control) and with 5, 10 µg/mL quinoxalone for 48 h. After washing with 75% ethanol, the cells were respectively incubated with anti-Bcl-2 antibody, anti-Bax antibody and anti-53 antibody. Then, the cells were incubated with FITC-conjugated secondary goat anti-mouse IgG. Bax (A), Bcl-2 (B) and p53 (C) levels were checked by flow cytometry.