IMAGE

Fig. 5

ID
ZDB-IMAGE-221220-50
Source
Figures for Pereira Sena et al., 2021
Image
Figure Caption

Fig. 5

Pharmacological inhibition of OGT reduces polyQ-expanded ataxin-3 levels and aggregates and increases autophagy. (A) Western blot analysis of protein extracts revealing that full-length Atx3 148Q (fl148Q, red arrowhead) was reduced upon OGT inhibition, while Atx3 15Q (fl15Q, blue arrowhead) remained unchanged. GAPDH served as loading control. n = 5 and one-sample t test; in 148Q DMSO versus 148Q OSMI-1, P = 0.044. (B) Filter retardation analysis of samples used in A, showing decreased aggregation of Atx3 148Q upon OSMI-1 treatment. n = 5, one-sample t test, and P = 0.010. (C) Cell-viability analysis of IHF derived from one control and one MJD patient demonstrating impaired viability of the MJD line, which was rescued by a 24 h treatment with 10 μM OSMI-1. n = 5, control DMSO versus control MJD, one-sample t test, and P = 0.021; in MJD DMSO versus MJD OSMI-1, paired t test and P = 0.0023. (D) Western blot for analysis of autophagy markers LC3B-II and p62 in 293T samples obtained after 10 μM OSMI-1 treatment plus 50 nM of the autophagy inhibitor BafA1 4 h before harvesting. Elevated LC3B-II and p62 levels upon OSMI-1 and BafA1 treatment suggest increased autophagic flux. β-actin served as loading control. - BafA1 = without BafA1; and + BafA1 = with BafA1. n = 3 and two-way ANOVA with Sidak’s post hoc test; in 10 μM OSMI-1 BafA1 versus DMSO BafA1, P = 0.0001 for p62 and P = 0.001 for LC3-II. (E) Western blot of iCN from MJD patients showing the reduction of global O-GlcNAc and of soluble ataxin-3 in samples treated with OSMI-1. Treatment of iCN derived from three MJD patients with 10 μM OSMI-1 for 24 h or 72 h, or 30 μM OSMI-1 for 24 h resulted in decreased polyQ-expanded ataxin-3 (Atx3 flexp, red arrowhead) in all three conditions compared to DMSO control (0 μM OSMI-1), whereas a significant reduction of WT Atx3 (Atx3 flwt, blue arrowhead) was only achieved with 30 μM OSMI-1 for 24 h GAPDH served as loading control. n = 3 and one-sample t test; in Atx3 flwt. 0 versus 30 μM OSMI-1, P = 0.0003; in Atx3 flexp 0 versus 10 μM OSMI-1 24 h, P = 0.0008; in Atx3 flexp 0 versus 10 μM OSMI-1 72 h, P = 0.016; and in Atx3 flexp 0 versus 30 μM OSMI-1 24 h, P = 0.003. (F) Filter retardation analysis of samples used in E revealing a reduction of ataxin-3 protein aggregates upon OSMI-1 treatment. n = 3 and one-sample t test; in 0 versus 10 μM OSMI-1 24 h, P = 0.043; in 0 versus 10 μM OSMI-1 72 h, P = 0.026; and in 0 versus 30 μM OSMI-1, P = 0.046. Data are represented as means ± SEM *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, and ns = not significant.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ Proc. Natl. Acad. Sci. USA