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

Figure 2

TGF-β1 induces NOX4 and ROS via Smad signal pathway in glioblastoma. (a) The correlation between TGF-β1 expression and NOX4 expression in glioma patients according to TCGA database and clinical specimens. (b) Immunofluorescent analysis of TGF-β1 (green) and NOX4 (red) coexpression in normal brain samples and GBM tissues.  μm. (c) Western blot analysis of the NOX4 protein expression levels with different concentrations of TGF-β1 (0, 1, 5, and 10 ng/ml) after 24 hours and time-dependent effects of TGF-β1 (10 ng/ml) treatment as evaluated for the glioblastoma cells. (d) Western blot analysis of the protein levels of N-cadherin, NOX4, Smad3, p-Smad3, Smad2, and p-Samd2 in glioblastoma cells treated with TGF-β1 in the presence or absence of SIS3 (10 μM) for 24 hours. (e) Western blot analysis for NOX4, p-Smad3, and p-Smad2 from the glioblastoma cells transfected with si-NC or si-Smad3 and then treated with TGF-β1 for 24 hours. (f) qPCR analysis of the NOX4 mRNA levels in glioblastoma cells treated with TGF-β1 in the presence or absence of SIS3 for 24 hours. (g) Glioblastoma cells were treated with TGF-β1 for 24 hours in the absence or presence of SIS3 (10 μM) and GKT137831 (10 μM) before staining for reactive oxygen species with CellROX Deep Red Reagents. ROS levels were calculated by the average fluorescent intensity.  μm. Data represent mean and SD of three independent experiments. ; ; .

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