Fig. 4

Hypoxia induces TFEB expression, and inhibition of TFEB suppresses mitophagy in HPAECs (A) Representative immunofluorescence images of single-labeling TFEB in control and hypoxia-treated HPAECs (n = 3 per group). Scale bar = 50 µm. (B) Quantitative comparison of the average optical density of TFEB staining among the groups (n = 5 per group). (C) Comparison of mRNA expression levels of TFEB, PINK1, and Parkin among the groups. HPAECs were transfected with siRNAs targeting TFEB, PINK1, or Parkin, along with a negative control siRNA (Thermo Fisher Scientific, USA), using Lipofectamine RNAiMAX transfection reagent (Thermo Fisher Scientific, USA). After transfection, cells were incubated under normoxia for 24 h, followed by hypoxia for additional 24 h before harvesting for analysis. (D) Flow cytometry analysis of mitochondrial membrane potential (ΔΨm) in each group (n = 3 per group). (E) Quantitative comparison of mitochondrial membrane potential (ΔΨm) among the groups. (F) Western blot analysis of TFEB and mitophagy-related proteins among the groups (n = 3). (G) Comparison of relative protein expression levels of TFEB and mitophagy-related proteins among the groups (n = 3). ns: not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.