Figure 4.

The ASAP is dependent on ROS and p38. (A) Quantification of IL-6 release from ECs treated with doxorubicin in the presence of RNA polymerase II inhibitor actinomycin D. n = 8. (B) IL-6 levels in media from doxorubicin- and/or cycloheximide-treated ECs. n = 7. (C) Flow cytometry plot of ECs stained with the ROS-sensitive dye DCF-DA after treatment with 225 nM doxorubicin. (D) EC IL-6 production. ECs were pretreated for 2 h with N-acetylcysteine (NAC) or glutathione (GSH) before the addition of doxorubicin. n ≥ 7 (E) IL-6 release from ECs treated with doxorubicin or the ROS inducers menadione or cumene hydroperoxide. n ≥ 5. (F) IL-6 secretion by ECs treated with doxorubicin in the presence or absence of the p38 inhibitor SB203580. n = 7. (G) IL-6 release by ECs treated with ROS inducers with or without the p38 inhibitor SB203580. n = 3. (H) Western blots of ECs treated with combinations of NAC and doxorubicin. Whole-cell lysates were collected 12 and 24 h after treatment. The intensity of phospho-p38 was quantified in ImageJ. Data are representative of two independent experiments. (I) IL-6 transcript levels from ECs treated with NAC and/or doxorubicin. RNA was extracted at 12 and 24 h after treatment. Transcript levels were measured by quantitative PCR and normalized to levels of GAPDH. For all IL-6 secretion measurements, IL-6 production over a 24-h period was determined by ELISA unless otherwise stated. Data are shown as mean ± SEM. (***) P < 0.001; (**) P < 0.01.