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Fig. 2. Expression of Tiam1 rescues the apoptotic sensitivity of Tiam1-KO keratinocytes. (A) Immunoblot showing the expression of exogenous full-length Tiam1 in Tiam1-KO cells. E-cadherin was used as loading control. (B) Quantification of apoptosis upon GF deprivation for 24 hours by annexin-V staining and FACS analysis of Tiam1-KO keratinocytes expressing empty vector or exogenous Tiam1. Error bars represent standard deviation from three independent experiments. (C) Phase-contrast photographs of Tiam1-KO keratinocytes expressing exogenous Tiam1 in GF-containing medium and following GF starvation for 24 hours. Bar, 50 µm. (D) GF starvation (24 hours) induced apoptosis in WT keratinocytes transfected with shRNA constructs and in Tiam1-KO cells. The luciferase targeting sequence was used as a control shRNA (sh-Luc). Tiam1 downregulation was approximately 50%. Apoptosis was monitored by PARP cleavage and was seen only in GF-starved cells expressing Tiam1-specific siRNA or Tiam1-KO cells. (E) Western blot showing keratinocyte apoptosis (PARP cleavage) induced by GF starvation in WT, Tiam1-KO, and Tiam1-KO with exogenous expression of either full-length Tiam1 or an inactive mutant of Tiam1 (Tiam1-
DH). Rac expression was used as loading control. (F) Estimation of apoptosis by quantification of free nucleosomes bound to DNA in WT, Tiam1-KO, and Tiam1-KO keratinocytes with exogenous expression of full-length Tiam1 or Tiam1-DH. Only the expression of full-length Tiam1 rescued apoptosis induced in Tiam1-KO keratinocytes by GF starvation. The presented data is a representative example of three independently performed experiments.
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