NOX4-Derived ROS Mediates TGF-β1-Induced Metabolic Reprogramming during Epithelial-Mesenchymal Transition through the PI3K/AKT/HIF-1α Pathway in Glioblastoma

Figure 3

NOX4-derived ROS mediates TGF-β1-induced metabolic reprogramming in glioblastoma cells. (a) Western blot analysis showed the time-dependent effects of metabolic enzymes under TGF-β1 treatment (10 ng/ml) on glioblastoma cells. (b) ROS generation was examined in normal glioblastoma cells transfected with sh-NC or shNOX4.  μm. (c) Western blot analysis for metabolic enzymes when NOX4 was suppressed or knocked down under TGF-β1 treatment on glioblastoma cells. (d) qPCR was conducted in control and shNOX4 glioblastoma cells to determine the mRNA levels of metabolic enzymes treated with TGF-β1. (e) Glioblastoma cells were treated with TGF-β1 for 24 hours before being adhered to microplates, and extracellular acidification rate (ECAR) was determined over time and analyzed as bar graphs. (f) Immunofluorescence images and (g) flow cytometry results of glucose uptake in glioblastoma cells determined by staining with 2-NBDG after treated with TGF-β1 in the absence or presence of GKT137831. Glucose uptake ability was calculated by the average fluorescent intensity.  μm. (h) Lactate production was determined in glioblastoma cells with or without NOX4 knockdown under treatment of TGF-β1 for 24 hours. (i) Glioblastoma cells were treated with TGF-β1 for 24 hours before being adhered to microplates, and oxygen consumption rate (OCR) was determined over time and analyzed as bar graphs. (j) Immunofluorescence microscopy of mitochondria in glioblastoma cells transfected with sh-NC or shNOX4 stimulated by TGF-β1 for 24 hours. The mitochondrial mass was visualized by MitoTracker staining and normalized by fluorescence.  μm. (k) Glioblastoma cells with or without NOX4 knockdown were treated with TGF-β1 for 24 hours before undergoing a PDH activity assay and western blot for the analysis of phosphorylated PDH. Data represent mean and SD of three independent experiments. ; ; .

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