Fig. 4

Effect on total body neutrophils from pik3cd^E1017K/+ and pik3cd^E525K/+ genotype and the PI3Kδ inhibitor CAL-101. (A) Light-sheet microscopy used to count total body TgBAC(mpx:gfp)i114 neutrophils of pik3cd^E1017K/+ and pik3cd^+/+ 3 dpf, sibling embryos. Upper panels show maximal-intensity projections for the three views used for each embryo. Scale bar: 100 µm. (B) Chart of total body neutrophils counted from light-sheet microscopy views as in A for 19 sibling 2 dpf pik3cd^E1017K/+_­and pik3cd^+/+ embryos. Bars show the mean±s.e.m., Mann–Whitney test. Data shown are from one experiment. (C) Chart of total body neutrophils counted from light-sheet microscopy views as in A, but for 21 pik3cd^E525K/+_­and pik3cd^+/+ 2 dpf sibling embryos. Bars show the mean±s.e.m., Mann–Whitney test. Data shown are from one experiment. (D) Composites of brightfield and the maximal-intensity projections of spinning disk confocal fluorescent (shown in green) micrograph stacks used to count TgBAC(mpx:gfp)i114 neutrophils in sibling pik3cd^E525K/+_­ and pik3cd^+/+ 3 dpf embryos. Scale bar: 100 µm. (E) Chart of neutrophils counted from single-sided maximal-intensity projections as in D for 96 sibling pik3cd^E525K/+_­ and pik3cd^+/+ 3 dpf embryos. Bars show the mean±s.e.m., Mann–Whitney test. Data shown are from one experiment. (F) Composites of brightfield and the maximal-intensity projections of spinning disk confocal fluorescent (shown in green) micrograph stacks used to count TgBAC(mpx:gfp)i114 neutrophils in sibling pik3cd^E1017K/+ and pik3cd^+/+ 2 dpf embryos exposed between 24 hpf and 48 hpf to either 25 µM CAL-101 in 1% DMSO or a 1% DMSO vehicle control. Scale bar: 100 µm. (G) Chart of neutrophils counted from single-sided maximal-intensity projections as in F for 95 sibling pik3cd^E1017K/+ and pik3cd^+/+ 2 dpf embryos exposed between 24 hpf and 48 hpf to either 25 µM CAL-101 or a DMSO vehicle control. Bars show the mean±s.e.m., multiple Mann–Whitney tests. Data shown are from one experiment. Two pilot experiments with CAL-101 also showed reduced neutrophils.