Fig 1: Metformin attenuates PM2.5-induced cell death and oxidative stress in BEAS-2B cells. Scramble shRNA (shScr)- and AMPKα2-specific shRNA (shAMPKα2)-stably transfected BEAS-2B cells were pretreated with PBS or 1 mM metformin for 2 h and then exposed to 50 μg/ml PM2.5 for 24 h. Cell viability (A) and intracellular ROS levels (B) were then determined. Lysates of the control and PM2.5-exposed shScr (C) or shAMPKα2 cells (D) with or without metformin pretreatment were examined by western blotting to determine the expression of phosphorylated AMPKα, SOD2, PRDX3, PRDX5 and TRXR2. β-Tubulin was used as a loading control. N = 3–4; data are presented as the mean ± SEM; * indicates p < 0.05, ** indicates p < 0.01, NS, not significant.
Fig 2: Metformin attenuates myocardial oxidative stress in PM2.5-exposed mice. After PM2.5 exposure, heart tissue was collected, and the myocardial 3′-nitrotyrosine (3′-NT) (A) and 4-HNE levels were measured. The heart sections were stained with Mitosox (scale bar = 20 μm), and the relative Mitosox fluorescence intensity was quantified (C, D). Lysates of the heart tissue were examined by western blotting for the expression of p-AMPKα, SOD2, PRDX5, PRDX3, TRXR2 and TRX2. β-Tubulin was used as a loading control (E, F). N = 4; data are presented as the mean ± SEM; * indicates p < 0.05, ** indicates p < 0.01.
Fig 3: Metformin ameliorates PM2.5-induced cell death and oxidative stress in H9C2 cells. H9C2 cells stably transfected with shScr or shAMPKα2 were pretreated with PBS or 1 mM metformin for 2 h and then exposed to 50 μg/ml PM2.5 for 24 h. Cell viability (A) and intracellular ROS levels (B) were then determined. Lysates of the control and the PM2.5-exposed shScr- or shAMPKα2-H9C2 cells with or without of metformin pretreatment were examined by western blotting to determine the expression levels of phosphorylated AMPKα, SOD2, PRDX3, PRDX5 and TRXR2. β-Tubulin was used as a loading control (C-D). N = 3–4, data are mean ± SEM; * indicates p < 0.05, ** indicates p < 0.01, NS, not significant.
Fig 4: Metformin attenuates PM2.5-induced pulmonary oxidative stress and cell death. After PM2.5 exposure, the levels of 3′-nitrotyrosine (3′-NT) (A) and 4-hydroxynonenal (4-HNE) (B) in lung tissue were measured. Lung sections from the control and PM2.5-exposed mice were stained with DAPI (blue) and TUNEL assay kit dye (red) (scale bar = 20 μm, arrows point to TUNEL-positive cells), and the TUNEL-positive cells were quantified (C, D). Lysates of lung tissue were examined by western blotting to determine the expression levels of total and phosphorylated AMPKα, superoxide dismutase 2 (SOD2), peroxiredoxin 3(PRDX3), PRDX5, thioredoxin reductase 2 (TRXR2) and Bcl-2. β-Tubulin was used as a loading control (E, F). N = 3–5; data are presented as the mean ± SEM; * indicates p < 0.05, ** indicates p < 0.01. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
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