Fig. 4

Autophagy induces HFSC glycolysis. A Representative immunofluorescence images of HFSCs treated with control, 3-MA(6 h) or Rapa(6 h) stained with anti-LC3B antibody(red) and anti-K15 antibody(green). B Representative immunofluorescence images of HFSCs treated with control,3-MA(6 h) or Rapa(6 h) stained with anti-P62 antibody(red) and anti-K15 antibody(green). C Quantification of LC3B-fluorescent dots/cell of control, 3-MA-treated or Rapa-treated HFSCs(up); Quantification of P62-fluorescent signal of control, 3-MA-treated or Rapa-treated HFSCs(down). D, E Protein extracted from HFSCs treated with Rapa (0.5 nM) for 6 h or treated with 3-MA (5 uM) for 24 h. Western blotting analysis of LC3B and P62 expression levels. (D) LC3B dots was evaluated 6 h post-administration with control, 3-MA or Rapa. E P62 expression was evaluated 24 h post-administration with control, 3-MA or Rapa. β-Actin was used as the loading control. F KEGG pathway enrichment analysis bubble chart. G Biological process category enrichment bubble chart of GO analysis. The Y-axis of the bubble chart represents GO or KEGG pathway terms, and the color of the bubble represents the P value of the terms. H, I Analysis of the concentrations of glucose and lactate in HFSCs supernatant cultured with control, 3-MA or different concentrations of Rapa for 18 h and 24 h. J Analysis of the concentration of lactate in HFSCs cultured with control, 3-MA or Rapa (0.5 nM) for 24 h. The data represent the means ± S.E.M. from at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, determined by Student’s t-test, 3-MA or different concentrations of Rapa versus Control