Fig. 5

Colocalization of emmprin/CD73 detected by immunofluorescent staining and in situ proximity ligation assay. Cytoplasmic CD73 (green) expression was observed in fibroblasts, tumor cells and co-culture cells. Membranous emmprin expression (red) was observed in tumor cells and co-cultured cells. Nuclei were stained with DAPI (blue). CD73 and emmprin were colocalized in tumor cells (b) and co-cultured cells (c). The green arrow points to fibroblasts expresssing green florescence (CD73); The yellow arrow points to tumor cells expressed yellow florescence (emmprin and CD73) (c). CD73 siRNA treatment causes downregulation of CD73 cytoplasmic expression, although, membranous emmprin expression was retained in co-cultured cells (d). The fluorescent red spots observed using in situ proximity ligation assay (PLA), indicating protein-protein colocalization in cells, confirmed the interaction between CD73 and emmprin. The detected dimers (emmprin/CD73) are represented as red dots in co-cultured cells (g). In cells transfected with CD73 siRNA prior to in situ PLA for emmmprin-CD73 interaction, CD73 siRNA treatment caused downregulation of red dots (h). Immunofluorescent staining, IF (a-d); In situ proximity ligation assay, PLA (e-h); fibroblast only (a, e); tumor cell only (b, f); co-culture (c-d, g-h)