Figure 3
Nanobody-induced reduction in EV release could be ascribed to CTTN or FSCN1 roles in endosomal maturation and/or invadopodia formation or maturation. (a) Representative confocal images of MDA-MB-231 breast cancer cells stably expressing ABP nanobodies (+dox, 24 h induction with 500 ng/ml dox) and without nanobody expression (−dox). Unlike EGFP and FSCN1 Nb5 expression, CTTN SH3 Nb2, CTTN NTA Nb2 and MOM-FSCN1 Nb5 expression induce a compaction of the Golgi apparatus (red). Extracted Golgi areas are shown in the insets. Nuclei (blue) were visualized by means of DAPI, Golgi apparatus (red) with anti-golgin-245 antibody and cell cortex (grey) by means of phalloidin staining. Nanobodies (green) are intrinsically fluorescent via their EGFP-tag. (b) Graphs represent quantification of Golgi spread area by means of golgin-245 staining in nanobody-expressing versus non-expressing MDA-MB-231 cells. Tukey boxplots represent quantification of the ratio of total golgin-245 area to total cell area. P0.05-value was determined by a Mann-Whitney rank sum test and n ≥100 cells were quantified for each boxplot. A compacted Golgi morphology can be observed in cells expressing (+dox) CTTN SH3 Nb2 (p = 0.0370), CTTN NTA Nb2 (p < 0.0001) and MOM-FSCN1 Nb5 (p = 0.0023), compared to their non-expressing (−dox) corresponding cell lines. Expression of EGFP as such has no effect on Golgi morphology (p = 0.2559), nor does expression of FSCN1 Nb5 (p = 0.5183). *P ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001. (c) Representative epifluorescence images of parental MDA-MB-231 cells seeded onto a polystyrene plate (48 h on serum-free medium). Invadopodia are visualized by immunofluorescence of CTTN and F-actin (Alexa fluor 594 phalloidin staining). Enrichment of both markers can be observed at the perinuclear region, which is typical of invadopodia. Boxed areas are enlarged in the bottom right insets and arrowheads Δ indicate areas of CTTN and phalloidin enrichment and colocalization.
