Rac1 Is Required for Epithelial Stem Cell Function during Dermal and Oral Mucosal Wound Healing but Not for Tissue Homeostasis in Mice
Figure 5
Impairment of human oral keratinocytes migration and proliferation after knock down of Rac1.
(A) The human oral keratinocyte cell line (NOK-SI) was transfected with siRNAs and knockdown of Rac1 was confirmed by Western blot analyses of Rac1 in cellular lysates 72 h after transfection of two different targeting siRNAs (siRNA#1 and siRNA#2). A non-targeting siRNA oligonucleotide (siControl) and untransfected cells (Control) were used as controls. Scratch wound assay in NOK-SI cell line after control and Rac1 siRNA#2. Scratches were generated after cell confluence. In vitro cell migration and wound closure were assessed every 12 h. Representative pictures of the control and Rac1 siRNA#2 transfected cell cultures at the indicated time after the initial scratch are depicted. (B) Top graphic, areas of migration were measured (dotted line from figure A) in multiple wells and represented at the indicated time. The lower graphic shows cell proliferation of NOK-SI transfected with siRNA#2 against Rac1. Incorporation of [3H]thymidine in cells stimulated with EGF (+) (30 ng/ml) or left untreated (-) was measured by the accumulation of radioactivity in cellular DNA, and represented as the average of CPM± s.e.m. in triplicate samples from a representative experiment that was repeated three times (***p<0.001; *p<0.05). (C) GST pull-down of active Rac1 was performed using NOK-SI stimulated with EGF. Cell lysates were incubated with GST-PAK-N for 30 min to affinity precipitate active Rac1. PAK-bound Rac1 and total Rac1 in the corresponding total lysates were analyzed by Western blotting with a monoclonal antibody against Rac1. Total and phospho-specific antibodies were used to detect two downstream targets of Rac1, JNK and PAK1 and their corresponding phosphorylated species. (D) NOK-SI cells were transfected with Rac1 siRNA (siRNA#2) or control siRNA (siControl) and stimulated with EGF (30 ng/ml). Western blot analysis of the Rac1 downstream targets JNK and PAK1 show absence of basal p-JNK and 3 min after EGF stimulation compared to control that show detectable basal levels of p-JNK that increase after EGF exposure. p-PAK1 also show reduced basal levels and a delayed accumulation after activation by EGF as compared to control cell lysates. GAPDH was used as loading controls. In C and D.