Fig 13. ROS promote actin clearance from lysosomes and actin depolymerization abates lysosomes coalescence during PIKfyve inhibition.

(A) RAW cells pre-labelled with Alexa⁴⁸⁸-conjugated dextran followed by treatment with vehicle, 20 nM apilimod for 40 min alone, or in presence of 10 μM CDNB for 30 min or 1 μM rotenone for 60 min. Cells were fixed with 4% PFA and stained for actin with phalloidin. Fluorescence micrographs were captured by spinning disc confocal as single z-planes. The inset is a magnified portion of field of view tracking Alexa⁴⁸⁸-conjugated dextran lysosome(s), phalloidin-stained actin, and as merged channels. Scale bar: 2 μm. (B) Cells were assessed for number of actin puncta structures associated with lysosomes. Data represent mean ± S.E.M. from three independent experiments, with 60–80 cells assessed per treatment condition across three experiments. One-way ANOVA and Tukey’s post-hoc test was used, where * indicates statistical significance between indicated conditions (p<0.05). (C) RAW cells were pre-labelled with Lucifer yellow and exposed to vehicle or 20 nM apilimod for 1 h, followed by apilimod removal at 0 or 2 h. These conditions were then supplemented with additional vehicle or 1 μM latrunculin A or 5 μM cytochalasin B for 1 h. Fluorescence micrographs are represented as z-projections of 45–55 z-plane images obtained by spinning disc confocal microscopy. Scale bar: 5 μm. D-F: Quantification of individual lysosome volume (D), lysosome number per cell (E), and total lysosome volume per cell (F). Data represent mean ± S.E.M. from three independent experiments, with 25–30 cells assessed per treatment condition per experiment. One-way ANOVA and Tukey’s post-hoc test was used, where * indicates statistical significance between indicated conditions (p<0.05).