Fig 2: FAK-mediated class IIa HDACs tyrosine phosphorylation is regulated by FFSS.a Immunoprecipitation by anti-phosphotyrosine (referred to as “p-Y-1000” throughout) antibody. FFSS was performed for 10 min with Ocy454 cells. Phosphotyrosine immunoprecipitation was performed on cell lysates treated as indicated, and protein expression was determined by western blotting. FFSS reduced HDAC4 and HDAC5 with p-Y-1000 immunoprecipitation. b FLAG immunoprecipitation was performed in HDAC5-deficient cells stably expressing FLAG-HDAC5, subjected to FFSS for times as indicated. phospho-HDAC5 was decreased in a time-dependent manner. c HEK293T cells were transfected with FLAG-HDAC4, then treated for one hour as indicated with PF562271 (10 µM) or vanadate (1 mM) followed by phosphotyrosine immunoprecipitation and then immunoblotting. Tyrosine-phosphorylated HDAC4 was increased by vanadate treatment and decreased by FAK inhibitor treatment. P-values adjusted for multiple comparisons vs control are shown. d FAK kinase activity was measured by ADP-Glo kinase assay. E4Y1 (poly(Glu)/poly(Tyr) ratio of 4:1) polypeptides were used as a control substrate of FAK tyrosine kinase. Increased FAK kinase activity was detected when recombinant human HDAC5 protein was used as a substrate. e Recombinant human HDAC5 and E4Y1 (a control substrate) were incubated with ɣ-32P-ATP and FAK tyrosine kinase for 1 h, then separate by SDS-PAGE followed by autoradiography. FAK treatment showed ɣ-32P-ATP-positive band at the expected HDAC5 recombinant protein size. f Kinase assay reactions as in e were separated by SDS-PAGE followed by immunoblotting as indicated. CBB indicates Coomassie Brilliant Blue stain. One-way ANOVA followed by Tukey–Kramer post hoc test was used (d). Data are expressed as mean ± SEM. Each experiment was repeated three times (a–c, e, f). Source data are provided as a Source Data file.

Fig 3: Immunohistochemical colocalization of HDAC3, HDAC4, and HDAC6 with neurons, astrocytes, and microglia in the ipsilateral superficial lumbar SCDH after CCI. Representative HDAC3 (A), HDAC4 (B), and HDAC6 (C) red immunofluorescence colocalized with NeuN (a neuronal-specific marker), GFAP (an astrocyte-specific marker), and OX-42 (a microglial cell–specific marker) green expression in sham and 3, 7, 14, and 28 days after CCI. The merged images (yellow) indicate colocalization of the HDACs with NeuN- and fewer GFAP-immunoreactive cells. Scale bar: 25 µm. CCI, chronic constriction injury; GFAP, glial fibrillary acidic protein; HDAC, histone deacetylase; SCDH, spinal cord dorsal horn.

Fig 4: Down-regulation of HDAC4 inhibits the migration of EC cells(A) Knockdown of HDAC4 reduces migration of KYSE30 and EC/CUHK1 cells. EC cells were transiently transfected with HDAC4 siRNA or a control siRNA. The number of migrated cells is decreased in cells (x100 magnification) transfected with HDAC4 siRNAs compared with those transfected with a control siRNA. Wound healing assay demonstrates that down-regulation of HDAC4 inhibits cell migration at 12 h and 24 h in (B) KYSE30 and (C) EC/CUHK1 cells (magnification, x100). (D) Western blot analysis indicates that knockdown of HDAC4 increases the expression of E-cadherin and α-catenin, and decreases the expression of Vimentin.

Fig 5: The conditional knockout of Hdac4 in cranial neural crest cells (CNCCs) results in an underdeveloped frontal bone. (A) Immunofluorescence staining of HDAC4 in frontal bone at E15.5. Western blotting of frontal bone and forelimbs in control and Hdac4fl/fl;Wnt1-Cre mutant mice at P0. (B) Posteroanterior, lateral, and frontal views of micro-computed tomographic rendering of a skull of a P0 Wnt1-Cre and Hdac4fl/fl;Wnt1-Cre mutant mouse. Note the decreased frontal bone formation in the Hdac4fl/fl;Wnt1-Cre mutants. The maxilla, mandible, palatine bone, and frontal bone were isolated at P0. Hdac4fl/fl;Wnt1-Cre mutant mice exhibited smaller frontal bones and there was a wider interfrontal gap between frontal bones compared with that in Wnt1-Cre (control) mice. The palatine bones of Hdac4fl/fl;Wnt1-Cre mutant mice were slightly smaller than those of Wnt1-Cre mice, while the maxilla and mandibles were of normal size and had no macroscopic deformities. (C) Quantification of the size (length, width, and height) of frontal bones from control (n = 3) and Hdac4fl/fl;Wnt1-Cre mutant mice (n = 3). Comparison of the surface area and volume of frontal bones from control (n = 3) and Hdac4fl/fl;Wnt1-Cre mutant mice (n = 3). P→A: posterior to anterior. *P < 0.05; **P < 0.01; ns. not significant. (D) Skeletal preparations showing the top view of the frontal bones in the P0 control and Hdac4fl/fl;Wnt1-Cre mutant pups. (E) Immunofluorescence staining of Ki67 and TUNEL staining in the frontal bone at E15.5.