Fig 2: CTS-induced HDAC4 nuclear relocation in chondrocytes.(A) Fluorescence microscope showed that GFP-HDAC4 was mainly located in the cytoplasm of cells in non-stretched control group (a-c), while GFP-HDAC4 was relocated to nucleus of cells subjected to CTS (d-f). Green indicated the GFP-HDAC4 and blue indicated cell nuclei stained by Hoechst 33342. (B) Percentage of GFP-HDAC4 located in nucleus was scored. 300 cells from 3 independent experiments were counted. Data were expressed as means±SD (P = 0.006). (C) Nuclear and cytoplasmic lysates were separated and followed by western blot analysis with anti-HDAC4 antibody. Histone 3 and GAPDH acted as loading controls for the nuclear and cytoplasmic fraction respectively (C-a). Semi-quantitative assay of band densities showed that cytoplasmic HDAC4 was decreased, and nuclear HDAC4 was increased in CTS group as compared to non-stretched control group (C-b), Values were presented as mean±SD (n = 3).

Fig 3: HDAC4, but not HDAC1 and HDAC5, rescues the defective IFN-α response in HDAC4−/− cells. (A) ISRE luciferase, Renilla, and HDAC4-FLAG expression plasmids were cotransfected into H4KO1 or H4KO2 cells (as indicated) overnight. The HDAC4-FLAG expression plasmid was transfected at 0, 10, 50, or 100 ng per well. Following overnight transfection, cells were stimulated with 1,000 units/mL IFN-α for 6 h. Cell lysates were prepared and firefly luciferase expression was measured and normalized to Renilla luciferase. Results show the fold induction of firefly luciferase relative to the unstimulated controls. (B) Reporter gene assay as in A performed with HDAC4 3SA-FLAG in H4KO1 or H4KO2 cells as indicated. (C) Reporter gene assay as in A performed with HDAC4 H803A-FLAG or HDAC4 D840N-FLAG in H4KO1 cells. (D and E) Reporter gene assay as in A but using HDAC5 (D) or HDAC1 (E). Data shown are representative of three independent experiments. Bottom shows immunoblots for FLAG-tagged proteins and α-tubulin (Tub). ns = not significant, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Fig 4: Kaplan-Meier survival analysis stratified according to a. HDAC-1, b. HDAC-2, c. HDAC-4 and d. HDAC-6 expression in pancreatic adenocarcinoma patients

Fig 5: Oxidation of HDAC4 upon Nox4 overexpression and treatment with H2O2. (A) BIAM switch redox assay (n = 5) (One-way ANOVA with Tukey post-hoc test, *p < 0.05), (B) relative mRNA expression analyzed by qPCR (n > 4) and (C) relative protein expression of HDAC4 analyzed by Western blot and normalized to ß-actin (n = 3) in Nox4 tet-on cells with or without tetracycline (1 µg/ml, 24 h) and DPI (3 µM, 3 h). (D) BIAM switch redox assay (n > 3) (One-way ANOVA with Dunnett post-hoc test, *p < 0.05) and (E) relative protein expression of HDAC4 in HUVECs with H2O2 (10/100/1000 µM, 30 min or 6 h) (n = 4). Representative Western blots are depicted. All bar graphs show means ± SEM.