Figure 5. Harmane induces autophagy in neuromelanin forming SH-SY5Y cells.

The galactose supplemented SH-SY5Y neuroblastoma cells were transfected with EV or TYR. After 48 h of transfection, cells were treated with 10 μM HAR or 10 μM PhIP for various time-points. A, B. The acridine orange assay was performed to assess lysosomal membrane permeability at various time-points from 1 h and 48 h post HAR and PhIP exposure, respectively. C, D. The LysoTracker™ green assay was performed to assess cellular lysosomal content at various time-points from 1 h and 48 h post HAR and PhIP exposure, respectively. ^ap<0.05, ^aaap<0.001 and ^aaaap<0.0001 compared EV non-treated control group; ^bp<0.05, ^bbp<0.01, ^bbbp<0.001 and ^bbbbp<0.0001 compared TYR non-treated control group; while, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 demonstrate significant difference between EV versus TYR-transfected SH-SY5Y cells at given concentration. E. Representative immunoblot showing expression of LAMP1, p62 and LC3B in SH-SY5Y cells transfected with EV or TYR constructs for 48 h followed by HAR (10 μM, 24 h) treatment. β actin was used as a loading control. F. The bar histogram represents the normalized densitometry of respective proteins. The data are represented as mean ± SEM, n = 3–4. *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 vs EV control group; ^ɸp<0.05 and ^{ɸ ɸ ɸ}p<0.05 vs TYR control cells; and ^{Δ Δ}p<0.01 and ^{Δ Δ Δ}p<0.001 vs EV HAR-treated cells.