Fig. 5

AMPK mediates the anti-fibrosis effects of amifostine. a Human fibroblast cells were transfected with nsRNA or siAMPK for 48 h prior to TGF-β1 and/or amifostine treatment. Fibronectin, α-SMA and Col1A1 levels were quantified by Western blot. Knockdown of AMPK was also confirmed by Western blot. n = 3, **p < 0.01, ***p < 0.001. The results shown are cropped from the original blots. b The effects of TGF-β1 and amifostine on AMPK activation were detected with the Thr172 phosphorylated AMPK antibody. The results shown are cropped from original blots. c Fibroblast cells were transfected with nsRNA or siAMPK for 48 h. The extracellular acidification rate was then measured as a reflection of cell metabolism. Cells were pretreated with TGF-β1 for 30 min, followed by amifostine treatment for another 30 min before the assay. Glucose (Gluc), rotenone (Rot) and 2-DG were added sequentially into the medium at 20 min intervals. The medium pH value was monitored and normalized to the cell lysate protein contents. n = 5, ***p < 0.001, ****p < 0.0001. d The oxygen consumption rate was measured by sequential addition of oligomycin (Oli), carbonyl cyanide-4 (trifluromethoxy) phenylhydrazone (FCCP) and rotenone + antimycin A (Rot). Data was normalized to the cell lysate protein contents. n = 5, **p < 0.01, ***p < 0.001