Research Paper Volume 12, Issue 24 pp 25581—25598

Figure 6. JMF3086 increased tubulin acetylation and kinesin-1 (kif5) expression. (A) Western blot of total and phosphorylated HDAC6, a-tubulin, acetyl-tubulin, and kinesin kif5 protein in stably transfected LRRK2-G2019S SH-SY5Y cells after treatment with different concentrations of JMF3086. (B) Quantification of the effects of JMF3086 on the ratio of acetyl-tubulin to total a-tubulin. The relative expression of acetyl-tubulin to total a-tubulin in SH-SY5Y controls, LRRK2-WT, and LRRK2-G2019S after treatment with DMSO solvent, 0.05 μM, 0.1 μM, and 0.5 μM JMF3086 was 0.19±0.02, 0.31±0.05, 0.27±0.03, 0.35±0.04, 0.56±0.08, 0.95±0.10, and 1.01±0.13, respectively. P=0.04 for LRRK2-G2019S with DMSO solvent vs. LRRK2-G2019S with 0.05 μM JMF3086; P=0.009 for LRRK2-G2019S with DMSO solvent vs. LRRK2-G2019S with 0.1 μM JMF3086; P=0.008 for LRRK2-G2019S with DMSO solvent vs. LRRK2-G2019S with 0.5 μM JMF3086, all one-way ANOVA. (C) Quantification of the effects of JMF3086 on Kif5 expression. The expression of Kif5 relative to GAPDH in SH-SY5Y controls, LRRK2-WT, and LRRK2-G2019S after treatment with DMSO solvent, 0.05 μM, 0.1 μM, and 0.5 μM JMF3086 was 0.66±0.05, 0.63±0.06, 0.54±0.04, 0.56±0.05, 0.79±0.08, 0.89±0.09, and 0.91±0.10, respectively. P=0.04 for LRRK2-G2019S with DMSO solvent vs. LRRK2-G2019S with 0.05 μM JMF3086; P=0.03 for LRRK2-G2019S with DMSO solvent vs. LRRK2-G2019S with 0.1 μM JMF3086; P=0.03 for LRRK2-G2019S with DMSO solvent vs. LRRK2-G2019S with 0.5 μM JMF3086, all one-way ANOVA. (D) Quantification of the effects of JMF3086 on the ratio of p-HDAC6 (Ser22) to total HDAC6. The relative expression of p-HDAC6 to total HDAC6 in SH-SY5Y controls, LRRK2-WT, and LRRK2-G2019S after treatment with DMSO solvent, 0.05 μM, 0.1 μM, and 0.5 μM JMF3086 was 0.31±0.05, 0.29±0.04, 0.31±0.03, 0.30±0.06, 0.29±0.05, 0.28±0.03, and 0.27±0.07, respectively. All experiments were repeated three times. Data represent mean ± SEM. *P<0.05, **P<0.01.