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. 2022 Oct 26;10:1015125. doi: 10.3389/fcell.2022.1015125

FIGURE 5.

FIGURE 5

Tau is hypoacetylated, a result of impaired production of acetyl-CoA upon Elongator depletion. (A) Western blotting to detect and quantify Acly, and ß-Actin in control and Elp1KD cells extracts. Histograms of proportion of Acly expression to ß-Actin. (B,C) Analysis of Acly activity by malate dehydrogenase coupled method performed in control and Elp1KD cells lysates. Histogram of relative Acly activity over assay (B) and of slopes (C) from the linear phase of the reaction. (D) Immunoprecipitation followed by Western blotting to detect and quantify Acetylated Tau, in control and Elp1KD cells extracts. Histograms of proportion of acetylated lysine expression to Tau. (E) Immunoprecipitation followed by Western blotting to detect and quantify acetylated Tau, in extracts from HEK293 cells transfected with Tau, Elp3 or empty vector. Histograms of proportion of acetylated lysine expression to Tau. (F) Western blotting to detect and quantify Hdac6, and ß-Actin in control and Elp1KD cells extracts. Histograms of proportion of Hdac6 expression to ß-Actin. (G) In vitro deacetylation assay of endogenously acetylated bovine brain tubulin incubated for 4 h with extracts of WT and Elp1KD cells, or without cells extract (control). (H) Immunoblotting to detect p300, ß-Actin in control and Elp1KD cells extracts and histograms of proportion of p300 expression to ß-Actin. (I) LC-MS quantification of Acetyl-CoA levels in control and Elp1KD cells lysates. All graphs show values of means ± SEM. Significance was determined by: (A–I) two-sided t test, Specifically, [(A) p = 0.0059; (C) p < 0.0001; (D) p = 0.0256; (E) p = 0.0019; (F) p = 0.9833; (H) p = 0.7225; (I) p = 0.0456; ] (G) two-sided Kruskal Wallis one-way ANOVA, Specifically, [p = 0.0003, K = 9.346; ] In addition, the post hoc multiple comparisons, to analyze statistical difference of each condition compared to control by Dunnett test, **p < 0.01. n = Number of experimental repeats: (A) Control n = 8; Elp1 KD n = 9; (C) Control n = 3; Elp1 KD n = 3; (D) Control n = 3; Elp1 KD n = 3; (E) Control n = 4; Elp1 KD n = 4; (F) Control n = 3; Elp1 KD n = 3; (G) Control n = 6; WT n = 3; Elp1 KD n = 3; (H) Control n = 4; Elp1 KD n = 4; (I) Control n = 3; Elp1 KD n = 3.