Fig. 6: A role of the MAP4K-HDAC6-TUBA4A axis in subcellular distribution of RANGAP1. | Cell Death & Disease

Fig. 6: A role of the MAP4K-HDAC6-TUBA4A axis in subcellular distribution of RANGAP1.

From: Screens in aging-relevant human ALS-motor neurons identify MAP4Ks as therapeutic targets for the disease

Fig. 6: A role of the MAP4K-HDAC6-TUBA4A axis in subcellular distribution of RANGAP1.The alternative text for this image may have been generated using AI.

A qRT-PCR analysis of shRNA-mediated knockdown of endogenous TUBA4A in human fibroblasts at 5 dpi (mean ± SEM; n = 3 biological repeats; ***p < 0.001, Student’s t-test). B Confocal images of RANGAP1 distribution in ALS1-hiMNs co-cultured with astrocytes at 28 dpi. Scale bar, 10 µm. C TUBA4A knockdown increases cytoplasmic fraction of RANGAP1 in ALS1-hiMNs at 28 dpi (mean ± SEM; n = 78 for shCtrl and n = 203 for shTUBA4A; *p < 0.05, Student’s t-test). D Western blots showing enhanced acetylation of TUBA4A (ac-TUBA4A) by Hit3 in ALS1-hiMNs at 21 dpi. E Confocal images of ac-TUBA4A in ALS1-hiMNs cocultured with astrocytes at 28 dpi. Scale bar, 10 µm. F Knockdown of MAP4Ks, comparable to Hit3 treatments, enhances TUBA4A acetylation in somas of ALS1-hiMNs (mean ± SEM; n = 78, 85 and 123 for the indicated groups, respectively; *p < 0.05, ordinary one-way ANOVA, when compared to shCtrl+Veh group). G qRT-PCR analysis of shRNA-mediated knockdown of endogenous HDAC6 in human fibroblasts at 5 dpi (mean ± SEM; n = 3 biological repeats; ****p < 0.0001, Student’s t-test). H Confocal images of ac-TUBA4A in ALS1-hiMNs cocultured with astrocytes at 28 dpi. Scale bar, 50 µm. I Confocal images of RANGAP1 distribution in ALS1-hiMNs cocultured with astrocytes at 28 dpi. Scale bar, 10 µm. J Knockdown of HDAC6 promotes TUBA4A acetylation in somas of ALS1-hiMNs at 28 dpi (mean ± SEM; n = 70, 71 and 69 for the indicated groups, respectively; ****p < 0.0001, ordinary one-way ANOVA, when compared to shCtrl+Veh group). K Knockdown of HDAC6 promotes nuclear localization of RANGAP1 in ALS1-hiMNs at 28 dpi (mean ± SEM; n = 84, 77 and 91 for the indicated groups, respectively; *p < 0.05 and ****p < 0.0001, ordinary one-way ANOVA, when compared to shCtrl+Veh group). L In vitro kinase assay and western blotting showing phosphorylation of purified GST-HDAC6 by HGK. M Confocal images of RANGAP1 distribution in ALS1-hiMNs cocultured with astrocytes at 28 dpi. Scale bar, 10 µm. N Knockdown of MAP4Ks, as well as Hit3 treatments, reduces hiMNs with HDAC6-induced abnormal cytoplasmic distribution of RANGAP1 at 28 dpi. O Knockdown of MAP4Ks, as well as Hit3 treatments, promotes nuclear localization of RANGAP1 even in the presence of HDAC6 at 28 dpi (mean ± SEM; n = 66, 56, 51 and 55 for the indicated groups, respectively; ****p < 0.0001, ordinary one-way ANOVA, when compared to shCtrl+Veh group).

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