Fig. 5
From: α-Synucleinopathy associated c-Abl activation causes p53-dependent autophagy impairment
Inhibition of c-Abl and p53 modulate basal autophagy, αS levels and regulate AMPK/mTOR signaling in neuronal cells. Hippocampal CLU neuronal cell lines (a) and M17 (A53T) human neuroblastoma cells (b, c) were treated with 2.5 μM nilotinib (Nilo) or 30 μM pifithrin-α (PFT) for 6 h (M17 cells) and 12 h (CLU cells). To determine autophagic flux, some cells were treated with 100 nM bafilomycin A1(BafA1) for the last 5 h. Following the treatments, the cell lysates were analyzed for LC3 and p62 (a, b) or pS129αS/total αS (c). α-Tub (α-tubulin) was used as a loading control. Data are means ±SEM from n = 3 separate experiments. a Analysis of BafA1 treated cells show that Nilo treatment leads to greater LC3-II levels following BafA1 treatment, indicating that c-Abl inhibition enhances basal autophagy. a,b Significantly different from c, p < 0.001, **p < 0.01; ***p < 0.001, One-way ANOVA followed by Tukey’s test. n = 3 per treatment. b, c Total lysates from M17 cells expressing human αS transgene were immunoblotted for LC3 and p62 (b) or pS129αS/total αS (c) following Nilo or PFT treatment. Quantitative analysis shows that both Nilo and PFT treatment increases basal LC3-II levels (b). Further, Nilo treatment reduces pS129αS levels (c). *p < 0.05; **p < 0.01; ***p < 0.001, Student’s t test. n = 3 per treatment. d, e, f, g Modulation of AMPK/mTOR signaling by c-Abl and p53 inhibition. Differentiated M17 (A53T) human neuroblastoma cells were treated with c-Abl inhibitor (2.5 μM Nilo, d and f) or p53 inhibitor (30 μM PFT, e and g) for 6 h. d, e Total lysates were immunoblotted for pS6, S6, p4EBP1, 4-EBP1. The data shows that Nilo and PFT treatment leads to reduction in the markers of mTOR activation. f, g Immunoblot analysis for pAMPK, AMPK, pS555ULK1 and ULK1. The data shows that Nilo and PFT leads to activation of AMPK and ULK1. Quantitative analysis of each figure was presented in bar graph. α-Tub (α-tubulin) was used as a loading control. Data are means ±SEM from n = 3. *p < 0.05; **p < 0.01; ***p < 0.001, Student’s t test