Fig 2: HDAC2 inhibition induced BRN3A expression in melanocytes and melanoma cells. (A) Basal protein expression of HDAC1, HDAC2, HDAC3, and HDAC11, in HM (3 donors), Hermes, and melanoma cell lines (1205Lu, WM165, WM1232, and WM35). (B) Protein expression of BRN3A and HDAC2 48 h after siRNA-mediated transient knockdown of HDAC2 in melanocytes (HM, Hermes) and melanoma cells (1205Lu). HDAC2 #1: HDAC2 siRNA. BRN3A induction upon HDAC2 inhibition was also confirmed by a second siRNA (HDAC2 #2, see Supplementary Figure S3A); control: non-targeting control siRNA; Ø: transfection reagent only (HM, Hermes: JetPrime; 1205Lu: Lipofectamine RNAiMAX). (C) Protein expression of BRN3A as well as HDAC1, HDAC3, and HDAC11 48 h after knockdown of HDAC1, 3, and 11, respectively, in melanocytes (HM, Hermes) and melanoma cells (1205Lu). HDAC1, HDAC3, and HDAC11: specific siRNAs targeting HDAC1, 3, and 11, respectively; control: non-targeting control siRNA; Ø: transfection reagent only (HM, Hermes: JetPrime; 1205Lu: Lipofectamine RNAiMAX). Representative immunoblots are shown.

Fig 3: ST8SIA6-AS1 upregulated HDAC11 via sponging miR-4656. a Analysis of TCGA data suggested that ST8SIA6-AS1 was positively correlated with HDAC11 expression in HCC. b Knockdown of ST8SIA6-AS1 decreased HDAC11 mRNA level in Huh7 and Hep3B cells. c Knockdown of ST8SIA6-AS1 decreased HDAC11 protein level in Huh7 and Hep3B cells. d miR-4656 mimic decreased HDAC11 mRNA level in Huh7 and Hep3B cells. e miR-4656 mimic decreased HDAC11 protein level in Huh7 and Hep3B cells. f pcDNA3-ST8SIA6-AS1 increased ST8SIA6-AS1 expression in Huh7 and Hep3B cells. g HDAC11 3′UTR-WT (wild type) and HDAC11 3′UTR-Mut (mutant) was inserted into luciferase plasmid. h, i Luciferase reporter assay indicated that miR-4656 mimic repressed luciferase activity of HDAC11 3′UTR-WT which was reversed by transfection of pcDNA3-ST8SIA6-AS1 in Huh7 (h) and Hep3B (i) cells. **p < 0.01; ***p < 0.001

Fig 4: S. aureus infection results in the downregulation of HDAC11 in macrophages, affecting the regulation of IL10 and mitophagy. (A) Heatmap of the top 10 differential protein expressions increased or decreased in BMDMs 24 h after intracellular infection with S. aureus. (B, C, and D) Western blotting analysis of HDAC11 and H3K9ac protein expression levels at various times after intracellular infection of S. aureus (MOI = 10). β-tubulin is used as a reference protein for quantifying HDAC11 expression, and H3 for H3K9ac. (E) ELISA quantitative analysis of IL10 levels in the supernatant of BMDMs infected with S. aureus (MOI = 10) at various time points. (F, G, H and I) Western blotting analysis of LC3, P62, mTOR, and p-mTOR protein expression levels at various time points during intracellular infection. GAPDH is used as a reference protein to quantify P62 expression; changes in LC3ii/LC3i and p-mTOR/mTOR are also quantified. (J, K, and L) Preparation of mitochondria (Mito) and Western blotting analysis of Pink-1 and Parkin protein expression levels at various time points during intracellular infection. COX IV is used as a reference protein to quantify Pink-1 and Parkin expression levels. Statistical analysis was performed using one-way ANOVA (C, D, G, H, I, K, L) or two-tailed Student’s unpaired t-test (E): ns, P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001. The data represent the mean ± SD from at least three independent experiments

Fig 5: HDAC11 affects mitophagy and intracellular mtROS clearance via mTOR. BMDMs were infected with S. aureus (MOI = 10) for 6 and 24 h, in conditions with or without overexpression of HDAC11 and treatment with Rapamycin (100nM). (A and B) JC-1 staining and flow cytometry analysis were used to assess changes in mitochondrial membrane potential. Quantitative analysis of the percentage of JC-1 monomer-positive cells. (C and D) Flow cytometry analysis of mtROS levels in BMDMs infected with S. aureus (MOI = 10) at various time points. Quantitative analysis based on Mean Fluorescence Intensity (MFI) indicating intracellular mtROS levels. (E) Confocal images show mitochondria (Mito-tracker, red), lysosomes (Lyso-tracker, green), and nuclei (Hochst, blue) within BMDMs (Scale bar: 10 μm). (F, G, H, and I) Western blot analysis of LC3, P62, mTOR, and p-mTOR protein expression levels in each group. GAPDH used as a reference protein for quantitative analysis of P62 expression levels and changes in LC3ii/LC3i, p-mTOR/mTOR ratios. (J, K, and L) Mitochondrial preparations were analyzed using western blotting to assess Pink-1 and Parkin protein expression levels in each group. COX IV was used as a reference protein to quantify the relative expression levels of Pink-1 and Parkin. Statistical analysis was performed using a two-tailed Student’s unpaired t-test: ns, P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001. The data represent the mean ± SD from at least three independent experiments