PIK-fyve (fab1) knockdown phenocopies Trpml deficiencies. Hemocytes expressing fab1 RNAi were cultured ex vivo to evaluate phagocytic processing and cytoskeleton dynamics. (A) Left: Representative image of a fab1-deficient hemocyte cultured with fluorescently labeled bacteria and LysoTracker probe. Scale bar: 5 µm. Right: Phagocytic processing was quantified as the percentage of hemocytes containing bacteria within at least two acidic vesicles after 1 h of culture (n = 3 independent cultures, n ≥ 20 hemocytes/condition). t test (*, P value < 0.05). (B) Hemocyte migration speed quantification (n = 3 independent experiments, n ≥ 2 animals/experiment). t test (*, P < 0.05). (C) Representative image of a larval hemocyte expressing LifeAct-Ruby and sqh-GFP, as well as fab1 RNAi. Scale bar: 5 µm (D) Left: Actin dynamics in response to MLSA1 treatment in fab1-deficient hemocytes. Data are shown as changes in area over time with respect to average (dashed line). Right: Actin dynamics quantified as SD of the area over time (n = 3 independent cultures, n ≥ 4 cells/condition, 30-min movies, 1-min intervals). One-way ANOVA with Tukey's multiple comparisons post hoc test; statistically equivalent values are represented with the same letter (P value < 0.05). Full videos are included in Video 4. The values in A are shown as scatter dot plot, indicating mean ± SD, and values in B and D as box and whiskers, where “+” indicates mean values.