Fig 2: HDAC4 accumulated in the nuclei of the fat body cells in AKHR mutants under prolonged fasting.(A) Immunohistochemical analyses of HDAC4 (anti-FLAG antibody, green) in AKHR mutant (AKHR 1) L3 larvae in 10 hr fasting condition as denoted in figures. Cell nuclei were stained by Hoechst 33258 (blue). Genotypes are as follows: Control (FB-Gal4,AKHR 1/AKHR 1) and FLAG-HDAC4,AKHR1 (FB-Gal4,AKHR 1/UAS-HDAC4,AKHR 1). The graphs showed the staining intensity profile for each antibody along the red lines. Scale bars, 20 μm. (B) Effects of the fat body-specific knockdown of HDAC4 (HDAC4 RNAi) on bmm gene expression in AKHR mutant adult flies (AKHR 1) in 10 hr fasting condition. Genotypes are as follows: Control (FB-Gal4/+), HDAC4 RNAi (FB-Gal4/+;UAS-HDAC4 RNAi/+), AKHR1 (FB-Gal4,AKHR 1/AKHR 1), and AKHR1,HDAC4 RNAi (FB-Gal4,AKHR 1/AKHR 1 ;UAS-HDAC4 RNAi/+). (C) Immunohistochemical analyses of HDAC4 (anti-FLAG antibody, green) in the fat body cells following expression of constitutively active (T196E) SIK3 in 10 hr fasting condition as denoted in figures. Genotypes are as follows: Control (FB-Gal4/+), FLAG-HDAC4 (FB-Gal4/UAS-HDAC4), and FLAG-HDAC4,SIK3TE (FB-Gal4/UAS-HDAC4;UAS-SIK3 T196E/+). The graphs showed the staining intensity profile for each antibody along the red lines. Scale bars, 20 μm. Data are presented as mean ± SEM (**P < 0.01; ***P < 0.001).

Fig 3: 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.

Fig 4: Sustained mTORC1 activation hampers HDAC4 signaling upon denervation. a, b Western blot analysis of total and phosphorylated HDAC4 in TA control (Ctrl) and TSCmKO (TSC) muscles after 1, 3, 7, 14 and 28 days of denervation (De). Quantification of HDAC4 protein levels is given in b; n = 4 (1, 3, 7d) and 3 (14, 28d) Ctrl, 3 TSCmKO mice. c Transcript levels of Hdac4 in TA innervated (In) muscle from control and TSCmKO mice, and after 1, 3, and 7 days of denervation. n = 3 per group. d, e Proportion of HDAC4-positive myonuclei in 3-day-denervated control and TSCmKO muscles (d), and the corresponding immunostaining showing HDAC4 (red) and laminin (green) in innervated muscle and after 3 to 28 days of denervation (e). Representative of 3–4 independent muscles per genotype. Arrows point to HDAC4-positive myonuclei. Scale bar, 50 µm. d, n = 3 per group. f Confocal pictures of endogenous HDAC4 (red) and lamin (green) in myonuclei from control and TSCmKO muscles after 3 and 28 days of denervation (from 4 (3d) and 3 (28d) independent muscles). Scale bar, 5 µm. 3D reconstruction is given in Supplementary Fig. 5a. g–o mRNA levels of HDAC4 targets Eno, Pfkm and Myh4 (g, h) of MyoG, Chrna1, Chrng, Myod1 and/or Myh2 (i–k), of the corepressors Mitr and Dach2 (l, m), and of Trim63, Fbxo32, Ctsl and Gabarapl1 (n, o) in TA innervated muscle and after 1 (i), 3 (g, j, l, n) and 7 (h, k, m, o) days of denervation in TSCmKO and control mice. Transcript levels are relative to Tbp mRNA and to Ctrl innervated muscle and, analyzed as the log2 fold change (FC) for (g, h, j, k, n, o). n = 3 per group. All values are mean ± s.e.m.; two-way ANOVA with Fisher’s (b) or Tukey’s (c, g–o) post-hoc tests, or two-tailed unpaired Student’s t-test (d), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Western blot quantifications are shown in Supplementary Table 1. Source Data are provided in the Source Data File

Fig 5: SIK inhibitors regulate SOST and RANKL expression.(a) Structure of YKL-04-114 (left), YKL-05-093 (middle) and YKL-05-093 Kd determination curves for SIK2 (right). For the Kd determination curves, the y-axis represents the amount of bound kinase measured by qPCR (see Methods), and the x-axis represents the corresponding compound concentration in nM. (b) Ocy454 cells were treated with YKL-04-114 (10 μM) for the indicated times, followed by immunoblotting of whole cell lysates as indicated. (c) Ocy454 cells were treated with the indicated concentrations of YKL-04-114 for 60 min, followed by immunoblotting of whole cell lysates as indicated. (d) Left: Ocy454 cells were treated with vehicle, PTH (50 nM) or YKL-05-093 (10 μM) for 60 min. Cytosol and nuclear fractions were then generated, followed by immunoblotting as indicated. Right: quantification of nuclear fraction (defined as nuclear/total) of HDAC4 or CRTC2. * indicates P<0.01 comparing treatment versus vehicle. (e) Top: Ocy454 cells were treated with the indicated concentrations of YKL-04-114 for 4 h, followed by RT-qPCR. Bottom: Cells were treated with YKL-04-114 (0.5 μM) for the indicated times. * indicates P<0.05 comparing treatment versus vehicle. (f) Cells lacking SIK2, SIK3 or both were treated with YKL-05-093 (10 μM for 45 min). Quantification of HDAC4 S246 phosphorylation, as assessed by densitometric analysis of immunoblots, is shown. * indicates P<0.01 comparing treatment versus vehicle. # indicates P<0.05 for the same comparison. (g) Control and SIK2/3 deficient cells were treated with YKL-05-093 (0.5 μM) for 4 h and SOST transcript abundance was measured by RT-qPCR. (h) Control and CRTC2 shRNA cells were treated with PTH (1 nM) or YKL-05-093 (0.5 μM) for 4 h and RANKL transcript abundance was measured by RT-qPCR. (i) Control and Gsα-deficient Ocy454 cells were treated with PTH (1 nM), YKL-05-093 (0.5 μM) or forskolin (5 μg ml−1) and SOST and RANKL transcript abundance was determined by RT-qPCR. For (h) and (i), * indicates P<0.05 comparing treatment and vehicle. (j) Control and Gsα-deficient Ocy454 cells were treated with PTH (50 nM), YKL-05-093 (10 μM) or forskolin (5 μg ml−1) for 30 min. Whole cell lysates were generated and immunoblotted as indicated.