Fig. 5: Retromer stabilization reduced the amount of insoluble aS and alleviates rotenone-induced cytotoxicity.

A SH-SY5Y cells preincubated for 4 days with a concentration of 5 μM R33, a retromer stabilizer, were sequentially solubilization with RIPA and urea buffer, and analyzed by WB. R33 treatment did not change the amount of RIPA-soluble aS but decreased the RIPA-insoluble, urea-soluble aS level. β-tubulin was used as the loading control. B Comparison of aS/β-tubulin signal ratio in the RIPA- and urea-soluble fractions. Values of the R33-treated cells are expressed relative to the values of the R33-untreated cells which are preincubated as 1.0, and the calculated values are expressed as the mean ± standard error. Data are statistically analyzed using the Student’s t test. *p < 0.05, n = 4. C SH-SY5Y cells before and after R33 treatment were sequentially solubilized with PBS-, Triton X-100-, and SDS-buffer, followed by WB analysis. Treatment with R33 increased the PBS-soluble aS levels and slightly, but significantly, decreased the amount of Triton-insoluble, SDS-soluble aS. D Comparison of the aS/β-tubulin signal ratio in PBS-, Triton X-100-, and SDS-soluble fractions. Values of the R33-treated cells are expressed relative to the values of R33-untreated cells which are presented as 1.0, and the calculated values are expressed as the mean ± standard error. Data were statistically analyzed using the Student’s t test. *p < 0.05, n = 4. E SH-SY5Y cells with and without R33 pretreatment were exposed to rotenone for 24 h, and cleaved caspase-3 expression levels were evaluated through WB analysis. The level of cleaved caspase-3 remained lower in the R33-pretreated cells than in the untreated cells. F Signal ratio of cleaved caspase-3/β-tubulin in the WB analysis after rotenone exposure. Rotenone exposure (0.2 μM) significantly increased the expression levels of cleaved caspase-3 compared to baseline of the R33 untreated group. However, in the R33 preincubated group, the cleaved caspase-3 expression level remained unchanged. Values of rotenone-treated cells are expressed relative to the values for the rotenone-untreated cells which are presented as 1.0, and the calculated values are expressed as the mean ± standard error. Data were statistically analyzed by one-way ANOVA followed by the Bonferroni post hoc test. *p < 0.05, n = 4. G SH-SY5Y cells with and without R33 pretreatment were exposed to rotenone and immunostained with cleaved caspase-3 antibody. Cleaved caspase-3 signal (green) induced by rotenone was hardly detected in the R33-treated cells. Nuclei were counterstained with TO-PRO-3 (cyan). Scale bar: 20 μm. H SH-SY5Y cells with and without R33 pretreatment were exposed to rotenone for 24 h and cell survival was confirmed using an MTT assay. R33-pretreatment significantly improved cell survival. Data were statistically analyzed using two-way ANOVA test and the post hoc Bonferroni test. Quantitative data are expressed as the mean ± standard error. *p < 0.05, n = 6.