Fig 2: Overexpression of HDAC4 promotes tumor growth and metastasis in NPC.A The protein expression levels of HDAC4 were determined in S26 and 6-10B cell lines stably expressing vector or HDAC4 by western blotting. GAPDH was used as a loading control. B The proliferation of the indicated cells in vitro was determined by CCK8 assays. The data are presented as the means ± SD of three independent experiments. Student’s t-test, **P < 0.01. C The colony formation ability of the indicated cells was determined. The images on the right are representative images. The data are presented as the means ± SD of three independent experiments. Student’s t-test, **P < 0.01. D, E Cell migration and invasion were determined in the indicated cells as described in the “Methods” section. The images on the right are representative images in (D) and (E). The data are presented as the means ± SD of three independent experiments. Student’s t-test, **P < 0.01. F The weight of orthotopic tumors after subcutaneous injection of 6-10B-vector and 6-10B HDAC4 cells. The data are presented as means ± SD. Student’s t-test, **P < 0.01. n represents the number of nude mice in each group. G The number of metastases in the lungs of mice 8 weeks after the tail vein injection of 6-10B-vector or 6-10B HDAC4 cells. The data are presented as means ± SD. Student’s t-test, ***P < 0.001. n represents the number of nude mice in each group. H The cell cycle was investigated in S26-HDAC4 and 6-10B-HDAC4 cells by flow cytometry. The data are presented as the means ± SD of three independent experiments. Student’s t-test, **P < 0.01. I, J The relative mRNA and protein levels of EMT-related genes were determined by qRT-PCR (left) and western blotting (right), respectively; GAPDH and β-actin were used as loading controls, respectively. The data are presented as the means ± SD of three independent experiments. Student’s t-test, *P < 0.05, **P < 0.01.

Fig 3: The effect of Hdac4 on the proliferation of MC3T3-E1 cells. (A) RT-qPCR analysis of Ki67 expression in cells treated with tasquinimod (Tasq) (10 μM) for 7 days. Cell proliferation assays were performed for control and tasquinimod-treated (10 μM) cells at 0, 24, 48, 72, 96, or 120 h (0–5 days). (B) Images and quantification of EdU staining (red) in MC3T3-E1 cells after treatment with 10 μM tasquinimod for 7 days. Nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. (C) RT-qPCR analysis of Hdac4 knockdown in MC3T3-E1 cells transfected with negative control (NC) or one of three Hdac4-specific shRNAs (n = 3). Western blotting of HDAC4 in control and shRNA3-transfected cells. RT-qPCR of Ki67 in control and Hdac4 knockdown cells. (D) Immunofluorescence staining of Ki67 in control and shRNA3-transfected cells. (E) Flow cytometric analysis of the effect of Hdac4 knockdown on the cell cycle. The bar graph represents the percentage distribution of control and Hdac4 knockdown cells in different phases of the cell cycle. The cell cycle distribution of control and Hdac4 knockdown cells is shown. Cell proliferation assays were performed in control and Hdac4 knockdown cells at 0, 24, 48, 72, 96, or 120 h after transfection (day 0–5). (F) Images and quantification of EdU staining (red) in control and Hdac4 knockdown cells. Nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 4: Effect of sepsis induced by LPS injection (10 mg/kg ip.) on (A) HDAC-4 and (B) Myogenin mRNA, (C) HDAC-4, and (D) Myogenin in rat diaphragm and gastrocnemius muscles, 24 h after a single injection of LPS or saline solution. mRNA was measured by PCR and proteins by Western blot. Representative Western blots are shown in (C,D (bottom)). The data represent mean ± standard error of the mean (SEM) for n = 6–8 rats/group. * p < 0.05 and ** p < 0.01 versus their respective control rats treated with saline, # p < 0.05 and ## p < 0.01 versus diaphragm of rats treated with LPS. Diaph: diaphragm, Gast: gastrocnemius.

Fig 5: Tasquinimod suppresses the HDAC4-induced promotion of cell proliferation in vitro.A–D Cell proliferation was analyzed in the indicated stable cell lines by CCK8 (A, B) and colony formation assays (C, D) after treatment with 5 μM tasquinimod or vehicle (DMSO) for 48 h. The images on the right (C, D) are representative images, and the images on the left (C, D) are the statistical results. The data are presented as the means ± SD of three independent experiments. Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001. NS: nonsignificant. E, F Cell cycle was investigated in the indicated cells via flow cytometry after treatment with 5 μM tasquinimod or DMSO for 48 h. The left images are representative images. Each colored area represents cells at different phases of the cell cycle: cells in the G1 phase (green area), S phase (yellow area), and G2/M phase (blue area). The right images are the statistical results. The data are represented as the means ± SD of three independent experiments. Student’s t-test. *P < 0.05, **P < 0.01. NS: nonsignificant. G The protein levels of cyclin D1 in the indicated cells were determined by western blotting after treatment with 5 μM tasquinimod or DMSO for 48 h. β-Actin was used as a loading control.