Figure 5.

GM treatment caused mitochondrial membrane potential damage, severe mitophagy and an increase of p53 in HEI-OC1, which could be reduced by co-treatment of NAC. (A) Mitochondrial membrane potential was marked by use of the sensitive probe Rhodamine 123, and detected through the measurement of fluorescence intensity. GM stimulated cells showed an increase of Rhodamine 123 fluorescence at 24 h, which could be reduced by use of NAC. *p < 0.05, **p < 0.01. (B) Mitochondria and lysosomes were labeled by Tom 20 (yellow-green fluorescence) and Lamp-1 (red fluorescence) respectively. Immunofluorescence staining revealed no colocalization of mitochondria and lysosomes in control group. However, more than 20 colocalized particles of mitochondria and lysosomes were observed around the nuclei 24 h after GM treatment, the color of which were orange because of the complete overlap of yellow-green and red (blue arrows). The co-treatment of GM and NAC led to an effective reduce of the number of mitophagy (white arrow). (C) Quantification analysis of mitophagy level revealed the mitophagy particles were induced by GM stimulus and inhibited by NAC co-treatment. **p < 0.01. (D) The treatment of GM induced high level of LC3B and p53, which could be cut down by co-treatment of NAC. (E) Western blotting analysis verified the results of immunofluorescence, we could see the increase of LC3B-II, p53 and cleaved-caspase 3 after 24 h of GM stimulus, which could be decreased by NAC co-treatment. *p < 0.05, **p < 0.01. (F) Cell viability analysis through CCK8 kit showed that there was no obvious difference among control, GM and NAC co-treatment groups.