Fig. 4

Torin1 treatment recovered the lysosomal function injured by morphine in BV2 cells. A The TEM analysis of ultrastructure of mitophagosomes (red arrowheads) and autolysosomes (yellow arrowheads). B, C The representative fluorescent intensity of LysoSensorTM Green DND189 (B) and quantitative analysis are shown in C as measured by flow cytometry (n = 9). D The mRNA expression of lysosome-related genes (ATP6V0D1, ATP6V0D2, LAPTM4A and LAMP1) as measured by qPCR analysis (n = 3–4). E, F The nuclear translocation of TFEB was measured by confocal microscopy analysis and the quantified results are shown in F (n = 25 random fields). G The activation of mTOR was measured by Western blots (n = 3). H Representative images of confocal microscopy analysis of LAMP1 and LC3B with or without Torin1 treatment in morphine-treated BV2 cells. Bar = 2 μm. I Quantitative analysis of overlap coefficients, lysosomal phagocytosis ratios of LC3B and LC3 + phagosomes in Fig. 3H (n > 30 cells). Data represent the mean ± SEM. Student's t-test or Mann–Whitney U test were used to measure significance between two groups (* p < 0.05, ** p < 0.01, *** p < 0.001 and ns p > 0.05)