Figure 3.

Metformin ameliorated iron overload and ferroptosis in PA-treated WRL68 cells

(A) Cell viability assessed with a CCK-8 assay after 24 h or 48 h of pretreatment with different concentrations of PA (0.1 mM–0.6 mM). Data are represented as mean ± SEM (n = 4 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗p < 0.05, vs. control group; ^#p < 0.05, vs. PA 0.1 mM group; ^$p < 0.05, vs. PA 0.2 mM group; ^&p < 0.05, vs. PA 0.3 mM group.

(B) Cell survival assessed by CCK-8 after 24 h of pretreatment with different concentrations of metformin (0.01 mM–25 mM) and PA (0.4 mM) for 24 h. Data are represented as mean ± SEM (n = 5 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗p < 0.05, vs. control group; ^#p < 0.05, vs. PA 0.4 mM group.

(C–F) Intracellular levels of TG, MDA, GSH, and T-SOD in WRL68 cells treated with PA (0.4 mM) without or with metformin (0.5 mM, 1.0 mM, and 2.5 mM) or AICAR (1.0 mM). Data are represented as mean ± SEM (n = 3 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗∗p < 0.01, vs. control group; ^#p < 0.05, ^##p < 0.01, vs. PA 0.4 mM group; ^&&p < 0.01, vs. PA + Metformin 0.5 mM group.

(G) Distribution of iron between cells and supernatants in WRL68 cells treated with PA (0.4 mM), FAC (0.1 mM) without or with metformin (2.5 mM). Data are represented as mean ± SEM (n = 3 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗∗p < 0.01, vs. control group; ^$$p < 0.05, vs. PA + FAC group.

(H) Representative images of Oil Red O staining of WRL68 cells treated with PA (0.4 mM) in the absence or presence of Fer-1 (4 μM) or metformin (2.5 mM) for 24 h (magnification, 200×), scale bar: 100 μm.

(I) Western blotting of AMPKα, P-AMPKα, and GPX4 after 24 h of PA (0.4 mM) treatment in the absence or presence of Fer-1 (4 μM) or metformin (2.5 mM) in WRL68 cells. GAPDH was used as the loading control.

(J) Cell viability assessed by CCK-8 after 24 h of pretreatment with erastin (20 μM) without or with metformin (2.5 mM). Data are represented as mean ± SEM (n = 3 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗∗p < 0.01, ^∗∗∗p < 0.001.

(K–M) Intracellular levels of MDA, GSH and T-SOD in WRL68 cells treated with erastin (20 μM) without or with metformin (2.5 mM). Data are represented as mean ± SEM (n = 3 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗∗p < 0.01, vs. control group; ^$$p < 0.01, vs. erastin group.

(N) Analysis of total iron, Fe²⁺and Fe³⁺ in pellets of WRL68 cells after erastin (20 μM) treatment for 24 h without or with metformin (2.5 mM). Data are represented as mean ± SEM (n = 3 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗∗p < 0.01, vs. control group; ^##p < 0.01, vs. erastin group.

(O) Total iron, Fe²⁺ and Fe³⁺ in supernatants of WRL68 cells after erastin (20 μM) treatment for 24 h without or with metformin (2.5 mM). Data are represented as mean ± SEM (n = 3 in each group). Differences among groups were assessed by one-way ANOVA analysis. ^∗∗p < 0.01, vs. control group. PA, palmitic acid; Met, metformin; TG, triglyceride; MDA, malondialdehyde; GSH, glutathione; T-SOD, total superoxide dismutase; Fer-1, ferrostatin-1.