Skip to main content
. 2023 Jan 7;9(1):e12820. doi: 10.1016/j.heliyon.2023.e12820

Figure 2.

Figure 2

Hypoxia upregulates levels of Beclin–1 and autophagy. (A) The relative expression of Beclin–1 mRNA in GBM cells (U87 and U251) after exposure to hypoxia for 0, 4, 8, 16, 24, 48, and 72 h. Hypoxia for 16 or 24 h induced temporary upregulation of Beclin–1 mRNA expression in cells. BECN1: Beclin–1. *P < 0.05 compared with control cells (hypoxia for 0h). (B) The protein expression of Beclin–1 was increased after exposure to hypoxia for both 16 and 24 h. Left panel: Western blot analysis of Beclin–1 protein. Right panel: Quantification of the relative protein expression of Beclin–1. β–Actin was amplified for internal normalization. Con: control. *P < 0.05 compared with control cells. (C) Cells were incubated under normoxia and hypoxia for 16 h, and Beclin–1 staining was then visualized using fluorescence microscopy to detect its protein expression and localization. (D) Hypoxia induced the conversion of LC3–I to LC3–II. Left panel: Western blot analysis of LC3–I and LC3–II protein. Right panel: Quantification of the protein LC3–II/I conversion. *P < 0.05 compared with control cells. (E) Granular LC3–GFP puncta aggregated as the hypoxia time increased. Cells were transfected with GFP–LC3 and incubated under hypoxia for 0, 4, 8, 16, and 24 h. GFP–LC3 staining was visualized using confocal laser scanning fluorescence microscopy. (F) Hypoxia induced the accumulation of autophagosomes. Cells were incubated under hypoxia for 8 and 16 h, and autophagosomes were visualized by transmission electron microscopy.