Figure 5.

BK regulates particle ingestion via lysosomal exocytosis. (A–C) Intracellular calcium chelation abolishes the enhanced phagocytosis by BK activation. Fast intracellular Ca²⁺ chelator BAPTA-AM but not the slower chelator EGTA-AM blocked the upregulated large beads ingestion induced by NS1619 in BMMs. BMMs were treated with NS1619 (20 μM) with or without EGTA-AM (10 μM) or BAPTA-AM (1 μM) for 30 min prior to phagocytosis assays and then incubated with opsonized 4.5 μm or 0.8 μm polystyrene beads or SRBCs at 37 °C for 60 min. Scale bars: 10 μm. (D,E) Transfection of cMyc-BK-GFP but not Lamp1-GFP rescued the defective ingestion of 4.5 μm beads in BK KO BMMs. This was blocked by Syt VII-DN overexpression. BMMs were transfected with identical amounts of cMyc-BK-GFP, Lamp1-GFP, Lamp1-mCherry and Syt VII-DN-mCherry, respectively. Scale bars: 10 μm. (F,G) Syt VII-DN markedly reduced the effect of NS1619 on large particle uptake in BMMs. BMMs were transfected with Lamp1-mCherry or Syt VII-DN-mCherry for 24 hours, and then incubated with IgG-opsonized 4.5 μm beads for 60 mins. NS1619 (20 μM) were applied 30 min prior to phagocytosis assays. For particle ingestion, experiments were repeated three times with triplicated samples each time for all conditions. Totally, 150–200 cells were counted for each condition.