Figure 10. KDELR stimulation by Bodipy-KDEL triggers the phosphorylation of ASAP1 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 pASAP1 (pTyr 782, green) and phalloidin (blue). Merged images of red, green and blue signals are also shown (Merge). pASAP1 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 pASAP1 spots at invadopodia. (B) KDELR stimulation increases ASAP1 to invadopodia. A375MM cells were treated as in A, fixed, and stained for ASAP1 (green) and phalloidin (blue). Merged images of red, green and blue signals are also shown (Merge). ASAP1 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 ASAP1 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. (D) Quantification of pASAP1 immunofluorescence levels at invadopodia. Data are means ±SEM of pASAP1 immunofluorescence per cell (% of control), from three independent experiments, with at least 50 cells quantified per experiment. (E) Quantification of ASAP1 immunofluorescence levels at invadopodia. Data are means ±SEM, as indicated for D. (C, D, E) ***p <0.001, **p <0.001 compared to vehicle treated cells (t-test).