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Figure 6


Fig. 6. Nox-dependent ROS production controls ERK phosphorylation and survival. (A) The levels of intracellular ROS were quantified by DFC fluorescence measurement in cell lysates of WT and Tiam1-KO keratinocytes. WT and Tiam1-KO keratinocytes were cultured in the presence or absence of GFs for 4 hours and treated with the Nox inhibitor DPI (15 mM) in the last 30 minutes. Measurements were conducted as described in Fig. 4B. (B) Quantification of intracellular ROS levels in WT and Tiam1-KO keratinocytes treated with or without the antioxidant NAC (10 mM). NAC treatment was carried out for 30 minutes prior to analysis. (C) Quantification of intracellular ROS levels in WT and Tiam1-KO keratinocytes treated with or without the Nox inhibitor DPI (15 mM) or Apocynin (APO, 10 mg/ml) in the last 30 minutes. (D) Western blot showing expression of various Nox components (Nox1-Mox1, p67-phox, p47-phox and p22-phox) in WT and Tiam1-KO cells, Rac expression serves as loading control. (E) Immunoblot showing ERK phosphorylation in WT keratinocytes in control or in GF-deprived conditions (4 hours), in the presence or absence of NAC (10 mM) or DPI (15 µM). Total ERK and Rac expression were used as loading controls. The data are a representative example of two independently performed experiments. (F) Western blot showing PARP cleavage in WT keratinocytes GF-starved for 24 hours or heat shocked (90 minutes at 43°C). Cells were treated with NAC or DPI at the start of the experiment. Rac expression serves as a loading control. The figure is a representative example of four independently performed experiments. (G) Model of the regulation of cellular fate by survival and pro-apoptotic signaling. An apoptotic stimulus (e.g. GF starvation) induces the activation of Bcl2 pro-apoptotic proteins. Tiam1 acts in the survival pathway by controlling Rac-dependent Nox-mediated ROS production and ERK activation. GFs may provide survival signals independent of Tiam1-Rac signaling, leading to ERK and/or Akt or NF{kappa}B activation.





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