Kir7.1 does not regulate cell polarity. (A) Representative time-lapse imaging of ASAP3 and mCherry-CAAX in fish neutrophils at CHT treated with VU590 using Tg(lyzC: ASAP3;lyzC: mCherry-CAAX). Ratiometric analysis (ASAP3/mCherry-CAAX) was performed to indicate a reduction in ASAP3 signal. Scale bars: 50 μm. Data representative of three independent experiments. (B) Representative time-lapse imaging of ASAP3 and mCherry-CAAX in fish neutrophils randomly migrating in the head mesenchyme using Tg(lyzC: ASAP3;lyzC: mCherry-CAAX). Ratiometric analysis (ASAP3/mCherry-CAAX) was performed. Scale bars: 10 μm. Image representative of 15 neutrophils. (C) Representative time-lapse images of Kir 7.1-GFP and mCherry-CAAX during spontaneous migration in the head mesenchyme or chemotaxis to LTB4. Relative fluorescent intensity along the indicated line was plotted. Blue line indicates the ratio of GFP/mCherry. Images representative of 15 neutrophils for each condition. (D) Representative ratiometric images of PHAKT-GFP and mCherry during neutrophil migration to LTB4 in zebrafish Tg(lyzC:mCherry;lyzC:PHART-GFP), Tg(lyzC:kcnj13-2A-mCherry;lyzC:PHART-GFP), or Tg(lyzC:kcnj13-Q153H-2A-mCherry;lyzC:PHAKT-GFP). The ratio indicates PHAKT-GFP/mCherry. White arrows indicate the direction of cell migration. Red arrows point to protrusions with a high accumulation of PHAKT. Scale bars: 10 μm. Image representatives of 15 neutrophils from each group. (E) Representative ratiometric images of GCamp6s and mCherry during neutrophils migration to LTB4 in zebrafish Tg(lyzC:mCherry;lyzC:GCamp6s), Tg(lyzC:kcnj13-2A-mCherry;lyzC: GCamp6s), or Tg(lyzC:kcnj13-Q153H-2A-mCherry;lyzC: GCamp6s). The ratio indicates GCamp6S/mCherry. White arrows indicate the direction of cell migration. Red arrows point to areas with high-calcium flux. Scale bars: 10 μm. Image representatives of 15 neutrophils from each group.