Fig. 5. ROS and cell-matrix adhesions stimulate ERK phosphorylation in the absence of GFs. (A) Western blot depicting that ERK phosphorylation in GF-starved WT keratinocytes is dependent on the presence of ROS and negatively correlates with increasing concentrations of antioxidant (NAC). The cells were starved for 3 hours in the presence of different concentrations of the ROS scavenger NAC. Total ERK expression was used as loading control. (B) Immunoblot showing PARP cleavage and ERK phosphorylation in WT keratinocytes. The cells were cultured in the presence or absence of GFs and NAC, as indicated. PARP cleavage correlates with prolonged (20 hours) GF-starvation combined with NAC treatment. Rac was used as a loading control. NAC was added at the beginning of the experiment. (C) Phase-contrast images of WT keratinocytes subjected to GF starvation and/or NAC treatment. The images correspond to the conditions in B. Bar, 50 µm. The data presented are representative examples of three independent experiments. (D) Western blot showing ERK phosphorylation in WT and Tiam1-KO cells in adhesive control, GF-starved (3 hours) and suspension conditions. Total ERK serves as a loading control. Results are a typical example of three independent experiments. (E) Quantification of ERK activity from D, normalized for total ERK expression; readouts corresponding to GF-supplemented conditions were set to 1.