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. 2021 Jan 15;11:1483. doi: 10.1038/s41598-020-79339-2

Figure 4.

Figure 4

Ultrastructural and biochemical evidences of autophagy in 3-NPA toxicity. Representative electron micrographs of 3-NPA cell model showed evidences of autophagy at various stages starting from formation of autophagosomes (A-i) and formation of autolysosomes (A-ii). Accumulation of multilamellar vesicles (A-iii), a characteristic feature of incomplete autophagy was also observed. Mn treatment did not show discernable morphological changes (A-iv). Whereas, MPP+ treatment caused cytoplasmic vacuolations along with the presence of abnormal mitochondria (arrow) (n = 3 experiments per group). Arrows represent the described pathological features [schematic was generated using Microsoft Paint (2004)]. (B,C) Co-treatment of 3-NPA model at 2 and 4 mM with chloroquine (0–15 µM) showed a dose and time dependent increase in cell death as shown by cell viability assay (n = 6 trials per experiment; *p < 0.05, ****p < 0.0001 compared to its respective untreated control; $$$$p  < 0.0001 compared to 3-NPA treated N27 cells at 2 mM and 4 mM; ns-not significant). (D–J) Standard autophagy markers depicting different stages of autophagy were examined in 3-NPA/Mn/MPP+ treated N27 cells. LC3 conversion that represents the autophagosomal mass, was maximum in 3-NPA treatment (D,E, corresponding complete blots in supplementary Figure 9i (3-NPA) and ii (Mn and MPP+)) which was confirmed by CQ cotreatment (F,G, corresponding complete blot in supplementary Figure 9iv). The LC3 western data for Mn and MPP+ is from a blot that is different from the 3-NPA blot (D). The β-actin western data for Mn and MPP+ is from a blot that is different from the 3-NPA blot (D). The status of autophagy upon 3-NPA treatment was further evaluated upon co-treatment with CQ (15 µM), followed by assessment of the levels of LC3-II conversion (F,G) and p62 (F,H, corresponding complete blot in supplementary Figure 10v). LC3 converiosn was noted in 3-NPA, CQ and 3NPA + CQ; however, the differences among the three groups was not significant (ns). On the other hand, p62 showed significant over-expression in 3-NPA + CQ treatment both in comparison with control and CQ treatment alone (n = 3 trial per experiment; *p < 0.05, **p < 0.01, ***p < 0.001; compared to untreated control; #p < 0.05, compared to CQ treatment alone; $$p < 0.01, compared to 3-NPA treatment alone). p62/SQSTM1 was also higher in MPP+ and 3-NPA treated cells (I,J, corresponding complete blot in supplementary Figure 10i) indicating possible incomplete autophagy (n = 3 trials per experiment; *p < 0.05, **p < 0.01). LAMP1, the lysosome marker showed significant increase in MPP+ and 3-NPA model (K,L, corresponding complete blot in supplementary Figure 11i). The western data of LC3 in CQ + 3-NPA samples (F), p62 (G), LAMP1 (I) are from 2 different blots. All blots were cut to retain only the region depicting the protein of interest.