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. 2022 Sep 17;57:102482. doi: 10.1016/j.redox.2022.102482

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© 2022 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Fig. 7 — The protein protein interaction between GPX4 and ProTalpha and the impact on apoptosis in the pancreatic acinar cells.

(A) A representative image (n = 3) of immunoprecipitation of ProTalpha from the lysates of pancreatic acinar cells of chicks (2-wk old, +Se + Vit. E) by anti-GPX4 antibody.

(B) Co-immunofluorescence staining of ProTalpha (the anti-ProTalpha antibody was labeled with a red fluorescent dye DyLight™ 594) and GPX4 (green) in pancreatic acinar cells after transfected with GPX4 (OE) 48 h. The merged image of yellow fluorescence represents the co-localization of ProTalpha and GPX4 in the acinar cells. Nuclei were labeled using Hoechest33258 staining. Scale = 5 μm.

(C) A representative image (n = 3) of immunoprecipitation of GPX4 from lysates of pancreases of chicks (2-wk old, +Se + Vit. E verse –Se + Vit. E) by anti-ProTalpha antibody.

(D) A representative image (n = 3) of immunoprecipitation of GPX4 from the lysates of pancreatic acinar cells transfected with the GPX4 (OE) or siGPX4 construct by anti-ProTalpha antibody. Control, lysates of acinar cells without transfection; OE, lysates of acinar cells transfected with pcDNA3.1/GPX4-EGFP and Entranster-H DNA transfection reagent; siGPX4-1, lysates of acinar cells transfected with GPX4-siRNA-1 and Lipofect AMINE 2000 transfection reagent.

(E) Effect of ProTalpha siRNA on relative mRNA abundance and protein levels of ProTalpha in pancreatic acinar cells at different time points. Data are means ± SE, n = 6.

(F) Effect of ProTalpha siRNA (48 h after transfection) on viability of pancreatic acinar cells treated with 0 μM, 50 μM, or 100 μM H₂O₂ for 6 h. Data are means ± SE, n = 6.

(G) Effect of ProTalpha siRNA (48 h after transfection) on protein levels of ProTalpha, pro-CASP 9, cleaved-CASP 9, pro-CASP 3, and cleaved-CASP 3 of pancreatic acinar cells treated with 50 μM H₂O₂ for 6 h. Data are means ± SE, n = 3.

(H) Effect of ProTalpha and/or GPX4 siRNA (48 h after transfection) on apoptosis of pancreatic acinar cells treated with 0 μM or 50 μM H₂O₂ for 6 h. Data are means ± SE, n = 6.

In A, C, D, E, F, G, and H, values within a given gene, protein, time, or dose of H₂O₂ differ (P < 0.05) without sharing a common superscript letter.

Abbreviations: ACTB, beta actin; cleaved-CASP 3, cleaved caspase 3; cleaved-CASP 9, cleaved caspase 9; Control, acinar cells without transfection; CYTC, cytochrome c; GPX4, glutathione peroxidase 4; Nc, acinar cells both transfected with negative control siRNA and Lipofect AMINE 2000 transfection reagent; ProTalpha, prothymosin alpha; pro-CASP 3, pro caspase 3; pro-CASP 9, pro caspase 9; siGPX4-1, lysates of pancreatic acinar cells both transfected with GPX4 small interfering RNA 1 and Lipofect AMINE 2000 transfection reagent; siProTalpha-1, lysates of pancreatic acinar cells both transfected with ProTalpha small interfering RNA 1 and Lipofect AMINE 2000 transfection reagent; siProTalpha-2, lysates of pancreatic acinar cells both transfected with ProTalpha small interfering RNA 2 and Lipofect AMINE 2000 transfection reagent; siProTalpha-2/siGPX4-1, lysates of pancreatic acinar cells transfected with ProTalpha small interfering RNA 2, GPX4 small interfering RNA 1 and Lipofect AMINE 2000 transfection reagent.