Figure 3
CQ and AQ ameliorated the effect of HG on mitochondrial biogenesis and fragmentation in hRPTCs. HKC-8 cells were incubated in 5–30 mM D-glucose for 24 hours and stained or harvested for analysis. CQ (200 μM) or AQ (10 μM) were treated for 1 hour before changing culture medium into 30 mM D-glucose to explore the effects of the drugs on hRPTCs subjected to HG conditions. (A) Cells were stained with Mitotracker (red) or anti-Tom20 antibody (green). Representative confocal fluorescence images of MitoTracker and Tom20 showing that increased numbers of functioning mitochondria were present in hRPTCs after treatment with CQ or AQ under HG conditions. (B) Cell lysate were immunoblotted with antibodies to Tom20 and GAPDH. Western blot analysis showed that Tom20 expression was increased in hRPTCs treated with CQ or AQ under HG conditions. Band intensities representing Tom20 and GAPDH expression levels were converted into densitometry using ImageJ software in the ratio of Tom20 to GAPDH. Results are means ± SEM. for experiments in triplicate. (C) Cell lysate were immunoblotted with anti-Drp1, anti-Mfn1 and anti-GAPDH antibody. Band intensities representing Mfn1 and Drp1 expression levels were converted into densitometry using ImageJ software in the ratio of Mfn1 to Drp1. A recovered Mfn1-to-Drp1 ratio was observed in hRPTCs treated with CQ or AQ and cultured under HG conditions. Results are means ± SEM. for experiments in triplicate. (**p < 0.01 vs 5 mM, #p < 0.05, ##p < 0.01 vs 30 mM).
