Fig. 7
From: Cordycepin prevents radiation ulcer by inhibiting cell senescence via NRF2 and AMPK in rodents
Cordycepin activates NRF2 by promoting autophagic degradation of Keap1. a Quantification of mRNA expression for NRF2 from cordycepin-treated fibroblasts for the indicated times. b Western blot analysis of NRF2 levels in the control and cordycepin-treated cells in the presence of 100 mg/mL CHX for the indicate time periods (left). Quantification of the NRF2 band intensity by ImageJ in the control and cordycepin-treated fibroblasts in the presence of 100 mg/mL CHX for the indicate time periods. NRF2 levels in the untreated cells were normalized to 1(right). c Western blot analysis of KEAP1 levels from cordycepin-treated fibroblasts for the indicated times (top). Densitometry quantified data of Keap1 to β-Actin expression ratios, represented as a fold change to non-stimulated cells (bottom). This test was repeated three times. Representative images were shown. d Representative immunofluorescence pictures of KEAP1 of the tongues from normal mice and irradiated mice (control, prevention, treatment) 10 days after irradiation dose. e–g Fibroblasts were incubated in the absence or presence of MG132 (10 μM), (CQ, 20 μM) or 3-methyladenine (3MA, 5 mM) for 4 h in advance and then were treated with 200 μM cordycepin for the indicated times (kept with MG132, CQ or 3MA), and total proteins were harvested for detection of NRF2, KEAP1 and LC3 by western blot. h Western blot analysis of Atg7, p62, LC3 levels from cordycepin-treated fibroblasts for the indicated times. i Fibroblasts were treated with 200 μM cordycepin observed for autophagy using transmission electron microscopy. Arrows represent autophagy vacuoles. j–k Fibroblasts were transfected with control siRNA (si-Ctl), siRNA against Atg7 (si-Atg7) or siRNA against p62 (si-p62), and then were treated with 200 μM cordycepin for the indicated times, and total proteins were harvested for detection of NRF2, Atg7 or p62, KEAP1 and LC3 by Western blot. Bars represent 50 μm (d). Data in a and c represent the means ± S.D. (n = 3, **P < 0.01; student’s t-test; ns not significant)
