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. 2019 Apr 24;13:148. doi: 10.3389/fncel.2019.00148

Figure 2.

Figure 2

Inhibition of GSK3β protected SH-SY5Y cells from 6-OHDA by decreasing SIRT2 phosphorylation. (A) SH-SY5Y cells were pretreated with different concentrations of SB216763 for 30 min and then treated with or without 100 μM 6-OHDA for 4 h. Cell viability was measured by the MTT assay (*p < 0.05, ****p < 0.0001, one-way ANOVA followed by Dunnett’s post hoc test). (B) Endogenous SIRT2 was immunoprecipitated from whole-cell lysates, and p-SIRT2 was detected by an anti-phosphoserine/threonine/tyrosine antibody (*p < 0.05, **p < 0.01, one-way ANOVA followed by Tukey’s post hoc test). (C) Western blot analysis showing the acetylation of α-tubulin (ace-tubulin) and total α-tubulin levels in SH-SY5Y cells treated with DMSO or SB216763 at the indicated concentrations (*p < 0.05, one-way ANOVA followed by Dunnett’s post hoc test). (D) SH-SY5Y cells were pretreated with DMSO, 10 μM SB216763 or 10 μM AGK2 for 30 min and then treated with or without 100 μM 6-OHDA for 4 h. Ace-tubulin and α-tubulin were detected by Western blotting (***p < 0.001, two-way ANOVA followed by Bonferroni’s post hoc test). (E) HEK293 cells were treated with DMSO, 10 μM SB216763 or 10 μM AGK2 for 30 min after transfection with PCMV-HA as a control or GSK3β-HA for 24 h. Ace-tubulin and α-tubulin were detected by Western blotting (***p < 0.001, two-way ANOVA followed by Bonferroni’s post hoc test). (F) HEK293 cells were cotransfected with GFP (or GFP-SIRT2) and PCMV-HA (or GSK3β-HA) for 24 h. Ace-tubulin and α-tubulin were detected by Western blotting (*p < 0.05, **p < 0.01, two-way ANOVA followed by Tukey’s post hoc test). (G) The level of the SIRT2 protein was downregulated in SH-SY5Y cells transfected with SIRT2 shRNA (shSIRT2-1 or shSIRT2-2) compared to cells transfected with the control (shNC). SIRT2 and GAPDH were detected by Western blotting. (H) A histogram representing the percentage of viable cells. Cells were transfected with shRNA for 48 h and then treated with 6-OHDA for 4 h. The percentage of viable cells was calculated after 24 h (*p < 0.05, **p < 0.01, ns, not statistically significant; one-way ANOVA followed by Tukey’s post hoc test). The values are expressed as the mean ± SD, n ≥ 3 for each group.