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. 2020 Apr 27;16(11):2001–2013. doi: 10.7150/ijbs.43825

Figure 2.

Figure 2

Nicotine induced autophagy impairment in NRVMs. (A) Transmission electron microscope (TEM) was used to determine the effect of nicotine on autophagy flux, and (Scale bar=2 μm) (B) Western blot was also performed to determine the autophagy marker LC3-II and its specific substrate p62 expression, BafA1 (100 nM) and Rap (10 μM) were used as negative and positive control respectively. (C) The effects on autophagy flux after combined treatment of nicotine with bafA1. (D) ADV-RFP-GFP-LC3 transfection was used to detect the nicotine-induced autophagy impairment. Representative immunofluorescence images of NRVMs expressing RFP-GFP-LC3 and treated with nicotine (100 μM), Rap, BafA1 or vehicle control for 24 hours. Representative of n = 3 experiments. (Scale bar, 20 μm) (E)The key determinant of autophagosome-lysosome fusion LAMP2 and lysosome marker LAMP1 were tested by Western blot. (****, p < 0.0001;***, p < 0.001; **, p < 0.01; *, p < 0.05 n = 3).