Hypoxia induces mitochondrial autophagy through translational regulation of lysosomal proteins.

<p><b>A</b>. HCT116 cells were exposed to hypoxia or normoxia for 24 h. The levels of lysosomal proteins glucosamine (N-acetyl)-6-sulfatase (GNS), prosaposin (PSAP), and tripeptidyl peptidase 1 (TPP1) were detected by immunoblot analysis. The levels of mRNA expression were analyzed by quantitative real-time RT-PCR. Detection of β-actin protein and mRNA served as loading controls. Bar graphs show relative protein (upper) and mRNA (lower) levels normalized to β-actin from at least three independent experiments (*p < 0.05). <b>B</b>. HCT116 cells were grown under hypoxia or normoxia for 24 h. Cells were stained with tetramethylrhodamine methyl ester (TMRM) and analyzed by flow cytometry to detect the membrane potential of mitochondria. Bar graphs show mean fluorescence intensity of TMRM from at least three independent experiments (**p < 0.01). <b>C</b>. HCT116 cells were exposed to hypoxia (H) or normoxia (N) for 24 h and 48 h. Total cell extracts were analyzed by immunoblotting with HIF-1α, ATPB, and β-actin antibodies. The protein level of ATPB relative to β-actin was quantified. Bar graphs show relative ATPB protein level normalized to β-actin from at least three independent experiments (*p < 0.05, **p < 0.01). <b>D</b>. HCT116 cells were transfected with the empty vector (pLKO.1) or the pLKO.1 vector expressing the indicated shRNAs (shPSAP and shLAMP2). At 2 days post-transfection, cells were treated with hypoxia (1% O₂) for 24 h. Immunoblotting was performed using antibodies against ATPB, β-actin, PSAP, and LAMP2. The protein level of ATPB relative to β-actin was quantified. Bar graphs show relative ATPB protein level normalized to β-actin from at least three independent experiments (**p < 0.01).