Fig. 6
hNSC-Exos enhanced mitophagy by activating the PINK1/Parkin pathway. A HT22 was infected with PINK1 gene knockout lentivirus, and then the expression of PINK1 protein in transfection groups was detected by WB analysis. The group with relatively obvious expression was selected to simulate the KO-PINK1 model for experiments. B WB analysis showed the expression of mitophagy-related proteins (PINK1, Parkin, Beclin, and LC3B), and the data were normalized to GAPDH protein expression. C The expression of fluorescence (red dots) and autophagosomes (yellow dots) were observed. The bottom left corner panel magnifies the boxed area in the top panel, scale bar = 50 μm. The percentage of autophagosomes& autolysosomes per cell was quantified by ImageJ. D Mito Tracker Red CMXRos and Annexin V-FITC were used to jointly detect mitochondrial membrane potential level (red) and apoptosis (green), scale bar = 20 μm. The ratio of red/green fluorescence was quantified by ImageJ. E Representative fluorescence image of MitoSOX (red) and the mean mitoSOX red fluorescence was quantified by ImageJ, scale bar = 20 μm. F Expression of apoptosis-related markers Bax, BCL2, Caspase 3, and Cleaved caspase 3, and data were normalized to GAPDH protein expression. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data were presented as mean ± SD from three biological replicates, each performed in triplicate. Experimental groups ('Control,' 'H2O2,' and 'hNSC-Exos’) are as described in Fig. 2. The “KO-PINK1” group comprised HT22 cells in which the PINK1 gene had been knocked out and were then exposed to 400 μM H2O2, and the "KO-PINK1 + hNSC-Exos” group consists of HT22 cells in which the PINK1 gene had been knocked out and were then exposed to 400 μM H2O2, followed by treatment with hNSC-Exos