Bag‐1L Protects against Cell Apoptosis in an In Vitro Model of Lung Ischemia‐Reperfusion Injury through the C‐Terminal “Bag” Domain

Figures

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  Figure 1. Detection of Bag-1L in each group after adenovirus infection for 48 h. A dramatic increase in Bag-1L (a) mRNA and (b) protein levels in both the Bag-1L and Bag-1L△C groups compared with the respective adenovirus control groups and the noninfection group. ∗p < 0.01 vs. the noninfection group; #p < 0.01 vs. the Bag-1L control group; ∆p < 0.01 vs. the Bag-1L△C control group.

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  Figure 2. Viability of A549 cells in different groups after hypoxia/reoxygenation for different durations. ∗p < 0.01 vs. the noninfection group; ∆p < 0.05 vs. the Bag-1L△C control group; #p < 0.05 vs. the Bag-1L control group; &p < 0.05 vs. the Bag-1L△C group.

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  Figure 3. Apoptosis of A549 cells after hypoxia/reoxygenation. After treatment with hypoxia/reoxygenation for 6 h, overexpression of Bag-1L significantly increased the late apoptosis of A549 cells. (a) Representative results of Annexin V-APC/PI staining of A549 cells exposed to hypoxia/reoxygenation for 6 h obtained via flow cytometry. Q3-UL necrosis, Q3-UR late apoptosis, Q3-LR early apoptosis, and Q3-LL viable cells. (b) Data analyses of late apoptotic cells in each group. ∗p < 0.01 vs. the noninfection group; #p < 0.01 vs. the Bag-1L control group; ∆p < 0.01 vs. the Bag-1L△C control group; £p < 0.01 vs. the Bag-1L△C group.

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  Figure 4. Measurement of LDH activity in the culture medium. ∗p < 0.01 vs. the noninfection group; #p < 0.01 vs. the Bag-1L control group; ∆p < 0.01 vs. the Bag-1L△C control group; &p < 0.01 vs. the Bag-1L△C group.

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  Figure 5. Overexpression of Bag-1L altered the cell cycle distribution of A549 cells in response to hypoxia/reoxygenation. (a) After infection for 48 h, there were no differences in each phase of the cell cycle between groups with overexpression of Bag-1L or Bag-1L△C. (b) Hypoxia/reoxygenation decreased significantly the percentage of cells in G0/G1 phase and increased the percentage of cells in S phase in the Bag-1L group. ∗p < 0.01 vs. the G0/G1 phase in the noninfection group; #p < 0.01 vs. the G0/G1 phase in the Bag-1L control group; ∆p < 0.01 vs. the G0/G1 phase in the Bag-1L△C control group; £p < 0.01 vs. the G0/G1 phase in the Bag-1L△C group; §p < 0.01 vs. the S phase in the noninfection group; ¥p < 0.01 vs. the S phase in the Bag-1L control group; ¢p < 0.01 vs. the S phase in the Bag-1L△C control group; Ωp < 0.01 vs. the S phase in the Bag-1L△C group.

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  Figure 6. Expression levels of Bag-1L, HSP70, Bcl-2, Bax, and cleaved caspase-3 upon hypoxia/reoxygenation. (a) Western blots results for HSP70, Bcl-2, Bax, and cleaved caspase-3. Relative protein expression according to the optical density ratios of (b) HSP70 and (c) Bcl-2/Bax relative to β-actin: ∗p < 0.01 vs. the noninfection group; #p < 0.01 vs. the Bag-1L control group; ∆p < 0.01 vs. the Bag-1L△C control group; &p < 0.01 vs. the Bag-1L△C group. (d) Optical density ratio for cleaved caspase-3 relative to β-actin: ∗p < 0.05 vs. the noninfection group; #p < 0.05 vs. the Bag-1L control group; ∆p < 0.05 vs. the Bag-1L△C control group; &p < 0.05 vs. the Bag-1L△C group.

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  Figure 7. Effects of Bag-1L on AKT, p-AKT, ERK, and p-ERK protein expression levels after exposure to hypoxia/reoxygenation for 6 h. (a) Western blot results for AKT, p-AKT, ERK, and p-ERK. (b) Relative protein expression based on the optical density ratio relative to that for β-actin. Data are presented as the ratio of p-AKT/total AKT. ∗p < 0.01 vs. the noninfection group; #p < 0.01 vs. the Bag-1L control group; ∆p < 0.01 vs. the Bag-1L△C control group; &p < 0.01 vs. the Bag-1L△C group. (c) Cell proliferation with addition of ly29004 (5 μmol/l): p < 0.01. (d) Ly294002 altered the cell cycle distribution. (e, f) Phosphorylation of AKT was inhibited by ly29004. Three independent experiments were performed. ∗p < 0.01 vs. the A549 group; #p < 0.01 vs. the A549+ly29004 group; ∆p < 0.01 vs. the Bag-1L+ly29004 group.