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Fig. 7. PTEN regulates actin polymerization but not cell orientation. (A) HL60 cells were untransfected (N.T.) or transfected with control or PTEN-specific siRNA. PTEN and tubulin (loading control) were analyzed by western blot. The graph at the right shows the quantification of PTEN estimated by densitometry using tubulin values for normalization. (B) The cells in panel A were analyzed for chemotaxis toward fMLP in transwells. Cells (2x105) were added to the upper chamber; the number of migrated cells in the lower well was determined by FACS and depicted as a percentage of migrating cells. (C) Jurkat cells were transfected with a bicistronic plasmid encoding PTEN or PTEN-C/S and GFP. PTEN and GFP expression were analyzed by western blot. Equal protein amounts of a GFP-transfected HL60 cell lysate were included in the western blot to compare relative amounts of endogenous (HL60) and ectopically expressed PTEN (Jurkat). (D) The cells (2x105) in panel C were analyzed for chemotaxis toward CXCL12 in transwell assays; GFP-positive cells in the lower well were determined by FACS; results are expressed as the percentage of migrating cells. (E) The cells in panel C were analyzed for chemotaxis toward CXCL12 as in Fig. 2A; dark-phase and green fluorescence were recorded at 23-second intervals. Migration parameters for 
20 cells/condition were determined with specific software. Representative paths of three cells are shown; average speed is shown of all cells analyzed. *, start point; 
, pipette tip. (F) CXCL12-induced actin polymerization was measured by FACS in mock, PTEN-, and PTEN-C/S-expressing cells described in panel C, using Alexa-fluor 633-phalloidin.
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