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. Author manuscript; available in PMC: 2024 Oct 6.
Published in final edited form as: Nat Aging. 2023 Dec 8;4(1):95–109. doi: 10.1038/s43587-023-00538-3

Fig. 4 |. Enhancing TFEB function by RCAN1 KD via its nuclear localization.

Fig. 4 |

a, Heatmap representation of open DARs with shRCAN1 (rescuing) and closed DARs with shCaN (detrimental) harboring TFEB binding motifs, compared with shCtrl. Motif analysis from ATAC-seq was from four independent HD-MSNs (HD.43, HD.40, HD.47, HD.45; three biologically independent samples each) (FDR < 0.05, FC ≥ 1.5). Top legend depicts representative motifs for TFEB binding sites. b, KEGG pathway enrichment analysis (top) of TFEB binding motif-containing genes associated with DARs in a. Integrative Genomics Viewer snapshots (bottom) showing peaks enriched in shRCAN1-HD-MSNs (red) and reduced in shCaN-HD-MSNs (blue) within RB1CC1 and MAPK1 in comparison with shCtrl (gray). c, Representative immunoblotting (left) and quantification (right) of the expression of phosphor-TFEB (Ser142) from three independent HD-MSNs (HD.43, HD.40, HD.47; n = 3) transduced with shCtrl, shRCAN1 or RCAN1. Phosphor-TFEB (Ser142): **P = 0.0095, *P = 0.0495; P62: shCtrl versus shRCAN1 *P = 0.0497, shRCAN1 versus shRCAN1&RCAN1 cDNA *P = 0.0496; RCAN1: shCtrl versus shRCAN1 **P = 0.0023, shRCAN1 versus shRCAN1&RCAN1 cDNA **P = 0.0034. d, Representative image (left) and quantification (right) of nuclear TFEB from three independent HD-MSNs (HD.43, HD.40, HD.47) transduced with TFEB WT, shRCAN1 or TFEB phosphor-mutant (S142/211A (SA)). Cells were immunostained with anti-TFEB and TUBB3 antibodies. An average of 130 cells each were counted from three or more randomly chosen fields (n = 6, 3, 3). Scale bars, 20 μm. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test in c and d; ****P < 0.0001, **P < 0.01, *P < 0.05 and mean ± s.e.m. (c,d). The sample size (n) corresponds to the number of biologically independent samples (c,d).