Fig 2: Impaired nuclear import of HDAC4 contributes to endplate defect in TSCmKO muscle. a–c Confocal pictures of HDAC4 (red, a), myogenin (red, b) and acetylated histone H4 (red, c) combined with α-bungarotoxin (Btx, green) in single fibers isolated from innervated and denervated (De) control (Ctrl) and TSCmKO muscles. Arrows and arrowheads show sub- and extra-synaptic (Syn) myonuclei, respectively. Representative of 4 independent muscles. Scale bar, 25 µm. d Confocal pictures of electroporated HDAC4 (red) and laminin (green) in innervated and denervated control and TSCmKO muscles. Representative of 4 independent muscles. Scale bar, 5 µm. 3D reconstruction is given in Supplementary Fig. 7d. e Confocal pictures of electroporated HDAC4 (red), α-bungarotoxin (Btx, green) and laminin (gray) on muscle section (upper panel) or in isolated fibers (bottom panel) from TSCmKO denervated muscle. Arrows point to sub-synaptic nuclei. Representative of 4 independent muscles. Scale bar, 10 µm. f, g Representative confocal pictures of “old” (green) and “new” (red) AChRs in TSCmKO denervated muscles (14d), electroporated with GFP or GFP-HDAC4 (gray). Scale bar, 50 µm. Turnover quantification is given in (g); values are mean ± s.e.m.; n = 6 GFP and 7 HDAC4 independent assays (number of mice electroporated; 136 and 50 fibers quantified, respectively); two-tailed unpaired Student’s t-test, ***p < 0.001. Source Data are provided in the Source Data File

Fig 3: B55a targets PP2A to HDAC5 for dephosphorylation. A, ARVM expressing GFP or GFP-HDAC5 were treated with 10 nmol/L isoproterenol (ISO) or vehicle control (CON) for 10 minutes. GFP/GFP-HDAC5-containing complexes were immunoprecipitated from cell lysates, separated by SDS-PAGE and the resulting Western blots probed for B55a, PP2AC, and PP2AA. Representative Western blots and grouped data from 4 independent experiments. Unpaired t tests. *P<0.05 vs CON. B, NRVM expressing GFP-HDAC5 were transfected with scrambled (Scr) or PPP2R2A (B55a) siRNAs for 48 hours prior to treatment with 1 µmol/L ISO or CON for 60 minutes. Representative Western blots and grouped data from 4 independent experiments. Two-way ANOVA following by Sidak's multiple comparisons tests. For B55a: *P<0.05 vs Scr within the same treatment group (CON/ISO). For S259: *P<0.05 vs CON within the same siRNA group (Scr/B55a). AdV indicates adenovirus; ARVM, adult rat ventricular myocytes; GFP, green fluorescent protein; HDAC5, histone deacetylase 5; NRVM, neonatal rat ventricular myocytes; PP2A, protein phosphatase 2A.

Fig 4: Activation of insulin receptor increases phosphorylation of SIK3 by Akt.(A) Immunoblot analyses showing the effect of 4 hr fasting and constitutively active insulin receptor (InRCA) on Thr196 phosphorylation of SIK3 protein in larvae (top three panels). Anti-phospho-Thr196 SIK3, -Myc (SIK3 protein), and -ß-tubulin (TUB) antibodies were used. Densitometry of phospho-Thr196 SIK3 bands (bottom panel). FB-Gal4 was used to drive transgene expression in the fat body. (B) Immunoblot analyses showing the effect of constitutively active insulin receptor (InRCA) on Akt-dependent phosphorylation of SIK3 protein in larvae (top four panels). The lysates were immunoprecipitated with an anti-Myc (SIK3 protein) antibody, and then immunoblotted with an anti-phospho-Akt substrate antibody. Densitometry of phospho-SIK3 bands (bottom panel). (C) A schematic model for LKB1 and SIK3 function to regulate lipid homeostasis in Drosophila fat body. LKB1 regulates the nucleocytoplasmic localization of HDAC4 via SIK3-dependent phosphorylation. Under feeding condition, DILPs-induced Akt activation leads to SIK3 activation, thereby inhibiting HDAC4 activity by phosphorylation. Under short-term fasting conditions, the AKH pathway inhibits the kinase activity of LKB1 in phosphorylating SIK3 Thr196 residue and controls SIK3 activity via PKA-dependent phosphorylation. Unphosphorylated and nuclear localized HDAC4 deacetylates and activates FOXO to increase bmm expression [19], thereby reducing lipid storage. AKH-independent signaling modulates the LKB1-SIK3-HDAC4 pathway to induce bmm expression when fasting is prolonged. Data are presented as mean ± SEM (*P < 0.05; **P < 0.01; NS, non-significant).

Fig 5: Coadministration of SIK2 inhibitor and olaparib increases ?-H2AX and decreases phosphorylation of HDAC4/5/7 in OVCAR8 and MDA-MB-231 tumor xenografts.Representative images of IHC with indicated antibodies, ?-H2AX (A) and p-HDAC4/5/7 (B), from mouse tumor tissues. Scale bar: 50 µm. Positive cells per 100 cancer cells were counted and 1-way ANOVA with Tukey’s multiple-comparison test were performed (NS, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). #1 indicates mouse #1 and #2 indicates mouse #2 from 1 experiment.