Figure 8. KDELR stimulation by Bodipy-KDEL triggers the phosphorylation of cortactin at invadopodia.

(A) A375MM cells were grown on rhodamine-conjugated crosslinked gelatine (red) for 16 h in the presence of BB94. Following BB94 wash-out, the cells were incubated for a further 3 h with the membrane permeant KDELR agonist Bodipy-KDEL (3 μM) or with vehicle alone (Vehicle) as a control. After fixing, the cells were stained for p-cortactin (Tyr 421 of cortactin, green) and phalloidin (blue). Merged images of red, green and blue signals are also shown (Merge). p-cortactin immunofluorescence overlapping the invadopodia are shown in the enlargements of the boxed regions (small right panels: red green and blue signals). White arrows point to p-cortactin spots at invadopodia. (B) KDELR stimulation increases cortactin to invadopodia. A375MM cells were treated as in A, fixed, and stained for cortactin (green) and phalloidin (blue). Merged images of red, green and blue signals are also shown (Merge). Cortactin immunofluorescence overlapping the invadopodia are shown in the enlargements of the boxed regions (small right panels: red green and blue signals). White arrows point to cortactin spots at invadopodia. (A, B) The images are representative of three independent experiments. Scale bars, 10 μm. (C) Quantification of the degradation area per cell. Data are degradation area per cell (% of control), as means ±SEM of three independent experiments, with at least 50 cells quantified per experiment. *** p<0.001, compared to vehicle cells (t-test). (D) Quantification of p-cortactin immunofluorescence at invadopodia. Data are means ±SEM of p-cortactin immunofluorescence per cell (% of control), from three independent experiments, with at least 50 cells quantified per experiment. *** p<0.001, compared to vehicle treated cells (t-test). (E) Quantification of cortactin immunofluorescence at invadopodia. Data are means ±SEM, as indicated for C. **p <0.01, compared to vehicle treated cells (t-test).