MANF Inhibits α-Synuclein Accumulation through Activation of Autophagic Pathways

Figure 4

MANF inhibits the accumulation of SNCA^WT in PD cellular model by macroautophagy activation. (a)–(d) Effects of MANF on the levels of SNCA^WT and macroautophagic-related protein expression. SNCA^WT SH-SY5Y cells were treated with MANF (250 and 500 ng/ml) and Dox (600 ng/ml) for 24 h (a, b) or 48 h (c, d), respectively; then, the protein levels of SNCA, LC3-I/II, Beclin-1, and P62 were detected by western blot analysis. (e, f) SNCA^WT SH-SY5Y cells were treated with autophagy inhibition CQ (10 μM) for 48 h, and cell lysates were immunoblotted by anti-LC3 and anti-Beclin-1 antibodies. (g, h) After SNCA^WT SH-SY5Y cells were treated with Lenti-mCherry-eGFP-LC3B plasmid for 48 h, MANF (500 ng/ml), Dox (600 ng/ml), and CQ (10 μM) were added, respectively, for another 48 h. The fluorescence of mCherry and eGFP was observed using fluorescence microscopy and quantified by Image Pro Plus software. Scale bar is 50 μm. (i, j) SNCA^WT SH-SY5Y cells were treated with MANF (500 ng/ml), Dox (600 ng/ml), and CQ (10 μM) for 48 h. The protein level of SNCA^WT was detected by western blot analysis. Data were expressed as from three independent experiments. ^#, ^##, and ^$ vs. control group; and vs. Dox-treated group; ^& vs. combined treatment with MANF and Dox group.

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