Figure 5.

Nitrite-mediated cytoprotection is dependent on AMPK activation. Cells were treated with or without nitrite (25 µmol/L; 21% O₂) in the presence or absence of mitoTEMPO (2 µmol/L; 30 min). (A) Western blot of phospho-AMPK (p-AMPK), total AMPK (t-AMPK) and β-actin, and quantitation of p-AMPK/t-AMPK/β-actin 1 h after nitrite removal. n = 4 for untreated and n = 3 for mitoTEMPO. (B) Cells were transfected with an empty vector (Vct) or siRNA against AMPKα1. Representative immunoblot of AMPKα1 expression 48 h after transfection. Cell death after H/R initiated 1 h (grey) after treatment with nitrite in cells transfected with siRNA to AMPKα1. Cell death measured after H/R initiated 1 h following nitrite treatment is also shown for control cells and those pre-treated with compound C (5 µmol/L; open bars) n = 4. (C) Western blot representing oxidized AMPK and blot quantitation after nitrite treatment in untreated cells and those co-treated with mitoTEMPO. Asterisks indicate P < 0.01, n = 4. (D) Representative calcein fluorescence traces and quantification of permeability transition pore opening in cells after H/R in the absence and presence of nitrite (50 µmol/L) as well as in the presence of compound C and nitrite. n = 4. Hashes indicate P < 0.05.