Figure 6.

PINK1 could protect HEI-OC1 cells from GM-induced damage by promoting autophagy and inhibiting the excessive activation of p53 pathway. (A) There were more morphological abnormal cells in PINK1-silenced group even without GM exposure (black arrows). Western blotting showed the decrease of PINK1 expression in HEI-OC1 treated with specific PINK1-siRNA in comparison with NC group. (B) PINK-siRNA group treated by GM for 24 h showed slightly higher Rhodamine 123 fluorescence but lower DCFH-DA fluorescence compared with NC group. (C) PINK1-interfered group showed obviously lesser number of parkin particles in contrast to NC group in response to GM. (D) Immunofluorescence staining revealed that PINK1-interfered group showed decreased LC3B (yellow arrows) but more p53 in HEI-OC1 cells (red arrows). (E) Total proteins were extracted for agarose gel electrophoresis. Analysis verified that lower PINK1 expression would lead to a decline of LC3B-II, but an increase of p53 and cleaved-caspase 3 by stimulus of GM. (F) The results of CCK8 showed that the relative cell viability of PINK1-interfered group declined to some degree in contrast to NC group. Four-hundred micromolar GM exposure could not cause obvious cell death in NC group but in PINK1-silenced group at 24 h. The co-treatment of autophagy inhibitor 3-MA could sharply decline the cell viability both in NC and PINK1-silenced groups. Inhibition of p53 pathway by use of PFTα could increase the cell viability of PINK1-interfered group in response to GM. Results were shown as mean ± SEM, *p < 0.05, **p < 0.01. (G) Flow cytometry analysis showed some degree of apoptosis in PINK1-siRNA group treated by GM, which was verified by TUNEL staining.