Fig. 7. Specific disruption of CME inhibits chemotaxis. (A) Tfn uptake in cells transfected with GFP, D32-GFP and DIII-GFP, separating the last condition into mock (–) and transfected (+) populations. For quantification, CME was considered totally inhibited when the cell lacked any cytoplasmic signal 20 minutes after Tfn addition. Averages ±s.d. are shown (n=100 cells by condition). (B) Cells transfected with DIII-GFP; and D32-GFP or GFP as controls, were allowed to migrate for 45 minutes in response to 100 ng/ml CXCL12 through transwell membranes. Chemotactic migration is analyzed as the ratio between migrated and non-migrated cells as a function of the fluorescence intensity levels, where region R0 corresponds to the non-transfected cells, and R1, R2, R3 and R4 correspond to increasing intensities. Migration ratios averages ±s.d. obtained from three (GFP), five (D32-GFP) and six (DIII-GFP) independent experiments are shown. Chemotactic analysis was performed only when CME inhibition was affecting >75% of DIII-GFP-transfected cells. The migration ratio of DIII-GFP significantly decreased as the fluorescence intensity (CME inhibition) increased (P<0.05). (C) Immunoblot of T cells transfected with control siRNA (siC) and siRNAs pre-designed against the clathrin heavy chain (si1, si2, si3) to calculate the efficiency relative to control. Note that only si2 and si3 significantly inhibited clathrin expression. (D) Tfn uptake quantification in siRNA-transfected cells. Note that CME is severely reduced but not completely abolished (n=50 cells by condition). (E,F) T cells transfected with siRNAs were allowed to migrate for 45 minutes in response to 100 ng/ml CXCL12 and for 1 hour in basal conditions through transwell membranes, respectively. The total number of migrated cells was quantified by flow-cytometry from three independent experiments, showing that the migration rates of si2 and si3 were significantly decreased compared with siC and si1 controls (P<0.05).