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. 2021 Sep 1;41(35):7479–7491. doi: 10.1523/JNEUROSCI.0476-21.2021

Figure 1.

Figure 1.

MAO-B inhibition facilitates α-syn secretion in SH-SY5Y cells. Western blottings of TCA-precipitated CM and cell lysates are labeled by Syn-1 antibody. β-Actin is used as a loading control. A, Parental SH-SY5Y cells were treated with selegiline for 24 h (n = 6). Cell lysates are blotted. Data were analyzed by Welch-ANOVA (F(3,20) = 1.272, p = 0.311) with Games–Howell post hoc test. B, CHX chase experiments of parental SH-SY5Y cells with and without selegiline. The intracellular levels of α-syn were analyzed by Western blotting of cell lysates with Syn-1 antibody (n = 9; F(4,40) = 26.140, p < 0.001, ANOVA). C, wt-aS/SH cells were treated with selegiline for 24 h. The extracellular levels of α-syn were analyzed by Western blotting of CM with Syn-1 antibody (n = 4; F(3,12) = 159.338, p < 0.001, ANOVA). D, Assessment of LDH release in selegiline-treated cells (n = 9). E, After treating wt-aS/SH cells with rasagiline for 24 h, CM and cell lysates were blotted (n = 6; F(3,20) = 14.391, p < 0.001, ANOVA). F, After treating wt-aS/SH cells with TVP-1022 for 24 h, CM and cell lysates were blotted (n = 4; F(3,12) = 0.546, p = 0.660, ANOVA). G, Assessment of LDH release in rasagiline-treated and TVP-1022-treated cells (n = 9). H, MAO-A or MAO-B knock-down wt-aS/SH cells were incubated in Opti-MEM for 24 h. The extracellular levels of α-syn were analyzed by Western blotting of CM with Syn-1 antibody (n = 6; F(2,15) = 7.394, p = 0.006, ANOVA). Left upper and middle panels show blots of cell lysates with MAO-A and MAO-B antibodies, respectively. Right upper and middle panels show blots of CM and cell lysates with Syn-1, respectively. Left lower graph shows cell viability in knock-down cells (n = 4). A–C, E, F, H, Graphs show the ratios of relative intensities of α-syn signals to controls. D, G, Graphs show LDH release ratios to lysis buffer-treated cells as a positive control. Data represent mean ± SD. B–H, Data were analyzed by one-way ANOVA with Bonferroni's post hoc test; *p < 0.05, **p < 0.01, ***p < 0.001. Control, Cont; cycloheximide, CHX; MAO-A, M-A; MAO-B, M-B; lysis buffer, Ly. buffer; rasagiline, Ras; secreted α-syn, Sec. αS; selegiline, Sel; TVP-1022, TVP.