3.2 XKP likely induced autophagy inβ-cells via mTO...
After XKP treatment for 36 h，the number of autophagosomes was significantly greater(Figure 2a，b)，the ratio of LC3-Ⅱ/LC3-Ⅰwas remarkably higher(Figure 2c)，and the P62 level was lower than the respective results of the control(Figure 2d).The ratio of LC3-Ⅱ/LC3-Ⅰin the XKP plus chloroquine treatment was higher than that in the XKP treatment(Figure 2f)，which indicated that XKP increased the autophagic flux rather than blocked fusion or degradation of autolysosomes.The phosphorylation of S6K，a substrate of mTOR，was lower，while phosphorylation of AMPK was unchanged(Figure 2e).These results indicate that XKP induces autophagy ofβ-cells via the mTOR pathway.
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Figure 2
(a)Cell immunofluorescence analysis.MIN-6 cells were treated with or without 1μL/mL of XKP extractive for 36h.Cells were stained with an anti-LC3 antibody and observed by confocal laser scanning microscopy.Scale bar，5μm.(b)Statistical results of cell immunofluorescence test.At least three replicates were performed.Error bars，s.d.；P＜0.01 compared to control cells by one-way ANOVA.The amount of autophagosomes per cell was quantified in at least 30 cells.c～fare western blotting analyses.MIN-6 cells were treated with or without 1μL/mL of XKP extractive for 36 h and then total cell extracts were immunoblotted with antibodies against(c)LC3 and GAPDH；(d)P62 and GAPDH；(e)S6K，p-S6K AMPK and p-AMPK；(f)LC3 turnover assay.MIN-6 cells were treated for 36 h with or without 1μL/mL of XKPextractive or with a combination of 1μL/mL of XKPextractive and 10μmol/L of chloroquine and then，total cell extracts were immunoblotted with antibodies against LC3 and GAPDH.