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Figure 3


Fig. 3. Tiam1-KO keratinocytes have impaired ERK phosphorylation – a necessary survival signal. (A) Quantification of apoptotic cells by annexin-V staining and subsequent FACS analysis. WT and Tiam1-KO keratinocytes were deprived of complete GF supplement and treated with EGF (20 ng/ml) or insulin (10 µg/ml) as indicated. Replacement of complete GF supplement by EGF or insulin rescued apoptosis. (B) Time-dependent induction of apoptosis and ERK1/2 phosphorylation upon GF starvation. Immunoblot shows PARP cleavage and ERK1/2 phosphorylation; Rac and total ERK are shown as loading controls. (C) Quantification of the data presented in B, normalized to total ERK2. (D) ERK signaling is required for survival upon GF starvation; insulin provides ERK1/2-independent survival signaling. WT and Tiam1-KO keratinocytes were cultured in absence of GF for 24 hours with or without EGF (20 ng/ml), insulin (10 µg/ml) or the MEK inhibitor PD-98059 (10 µM). The immunoblots shown are representative examples of three independent experiments. (E) Quantification of ERK1/2 phosphorylation from three independent experiments with conditions as in D. Values were normalized for total ERK and readouts corresponding to GF-supplemented conditions were set to 1. Error bars represent standard deviation. (F) Quantification of PARP cleaved product from three independent experiments with conditions as in D, values were normalized for total ERK. Error bars represent standard deviation. (G) Quantification of apoptosis induced by GF starvation, by annexin-V staining and FACS analysis. As for the experiment shown in D, WT and Tiam1-KO keratinocytes were cultured in absence of GFs for 24 hours with or without EGF (20 ng/ml), insulin (10 µg/ml) or the MEK inhibitor PD-98059 (10 µM). Error bars represent standard deviation from three independent experiments. (H) Western blot depicting phosphorylation of Akt and I{kappa}B in WT and Tiam1-KO keratinocytes in control and GF-deprived conditions (4 hours). Total Akt and Rac are presented as loading controls.





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