Fig. 4. Tiam1-KO keratinocytes have impaired ROS production. (A) The level of intracellular ROS in WT and Tiam1-KO keratinocytes was visualized by DCF staining. The cells were starved of GFs for 4 hours. Images were taken using an epifluorescence microscope. Bar, 50 µm. (B) Quantification of intracellular ROS in WT and Tiam1-KO keratinocytes upon GF starvation for 30 minutes and 4 hours. Cells were loaded with DCF, lysed and DCF-fluorescence was measured in the lysates. Fluorescence values were normalized for the amount of total protein present in the lysates. The fluorescence value of WT cells was set to 1. Error bars represent standard deviation from triplicate measurements. The result shown is a representative example of four independent experiments. (C) Quantification of ROS levels at a single-cell level in WT and Tiam1-KO keratinocytes by DCF staining and FACS analysis. (D) Western blot showing the exogenous expression of full-length Tiam1 and the deletion mutant of Tiam1 (Tiam1-DH) in Tiam1-KO keratinocytes. Expression of Rac is shown as a loading control. (E) Exogenous expression of full-length Tiam1 but not Tiam1-DH restores ROS production in Tiam1-KO keratinocytes. Intracellular ROS was measured as described in B. (F) Partial downregulation of Tiam1 reduces ROS production in WT keratinocytes, both in the presence and absence of GFs. Intracellular ROS content was measured as described in B. The data is a representative example of three independent experiments. (G) Immunoblot showing Tiam1 expression in WT keratinocytes upon shRNA expression. Partial downregulation of Tiam1 (50%) inhibits ERK1/2 phosphorylation (in GF-starved conditions) when compared with control cells. Tiam1 and total ERK were used as expression and loading controls, respectively.