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. 2011 Jan 31;6(1):e16571. doi: 10.1371/journal.pone.0016571

Figure 5. Silencing of Rac1 and IQGAP1 decreases HPAEC migration in an in vitro scratch assay.

Figure 5

HPAECs (∼50% confluence) grown on 35-mm dishes or on cover slips were transfected with either scrambled siRNA or Rac1 siRNA or IQGAP1 siRNA (50 nM, 72 h), then starved for 3 h in 0.1% FBS in EBM-2 without growth factors. In A, cells on cover slips were stimulated with 0.1% BSA-complexed S1P (1.0 µM) for 5 min, washed, fixed, permeabilized, probed with anti-Rac1 antibody and anti-IQGAP1 antibody, and examined by immunofluorescence microscopy using a ×60 oil objective. In B, HPAECs (∼50% confluence) were transfected with either scrambled siRNA or Rac1 siRNA or IQGAP1 siRNA (50 nM, 72 h) prior to scratching the cells for migration assay. Scratched cells were challenged with S1P (1.0 µM) for 16 h. The values are means ± S.E.M. for three independent experiments each done in triplicates. C - Cell lysates (20 µg total proteins) were subjected to SDS-PAGE and Western blotted with anti-Rac1 and anti-IQGAP1 antibody as described under “Experimental procedures”. Western blot is representative of three independent experiments.