Fig. 7

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