Figure 3.

V-ATPase inhibition synergized with CD47 blockades to induce PrCR. (A) Phagocytosis assays with BMDMs showing that pharmacological inhibition of V-ATPase with concanamycin A (10 nM) promoted PrCR and increased CD47-blocking antibody-induced phagocytosis. The assays were performed with human lymphoma (U973 and Raji), colon cancer (SW620 and DLD1) and breast cancer (MDA-MB-231) lines. Each group was compared with the Ctrl group. *P < 0.05, **P < 0.01 (one-way ANOVA test). (B) A phagocytosis assay with human peripheral blood monocyte-derived macrophages showing that concanamycin A treatment enhanced PrCR of SW620 and MDA-MB-231 cells in the absence or presence of CD47 blocking antibodies. Each group was compared with corresponding non-treated or treated Ctrl group. **P < 0.01 (one-way ANOVA test). (C) Representative flow cytometry plots for (B) Macrophages were labeled with anti-CD14. SW620 cells were GFP+. Concanamycin A treatment increased the percentages of human macrophage phagocytosing cancer cells (hCD14+GFP+) with or without anti-CD47 antibody. (D) An overnight phagocytosis assay showing macrophage-mediated PrCR induced by either CD47 blocking antibody or genetic deletion of CD47 was largely potentiated by treatment of SW620 and MDA-MB-231 cells with concanamycin A (the values of Ctrl^KD were normalized as 100%). **P < 0.01 (one-way ANOVA test). (E) A phagocytosis assay with BMDMs showing that genetic knockdown of ATP6AP2 expression was able to induce PrCR of SW620, DLD1 and MDA-MB-231 cells and synergized with knockdown of CD47 expression. Each group was compared with the Ctrl^KD group. *P < 0.05, **P < 0.01 (one-way ANOVA test). (F) A phagocytosis assay with BMDMs showing exogenous expression of ATP6AP2 inhibited PrCR of SW620 cells and reversed the enhanced PrCR of ATP6AP2^KD cells. *P < 0.05, **P < 0.01 (t test).