Fig 5. Apilimod integrity and PtdIns(3,5)P₂ levels are not altered by ROS.

(A) RAW cells pre-labelled with Lucifer yellow. Following reactions were performed in complete media in vitro for designated time, prior to adding to cells for an additional 40 min: vehicle; 1 mM H₂O₂ 40 min; 20 nM apilimod 40 min; 20 nM apilimod preincubated with 1 mM H₂O₂ for 40 min; 20 nM apilimod preincubated with 0.5 mg/L catalase for 60 min; 1 mM H₂O₂ exposed to 0.5 mg/L catalase for 60 min to neutralize H₂O₂, followed by 20 nM apilimod 40 min; or 20 nM apilimod exposed to 1 mM H₂O₂ for 40 min to test whether H₂O₂ degraded apilimod, followed by 0.5 mg/L catalase for 60 min to degrade H₂O₂. Fluorescence micrographs are spinning disc microscopy images with 45–55 z-planes represented as z-projections. Scale bar: 5 μm. (B-D) Quantification of individual lysosome volume (B), lysosome number per cell (C), and total lysosome volume per cell (D). AP (apilimod), CAT (catalase). Data are shown as mean ± s.e.m. from three independent experiments, with 25–30 cell assessed per treatment condition per experiment. One-way ANOVA and Tukey’s post-hoc test used for B-D; * indicates statistical difference against control condition (P<0.05). (E-F) ³H-myo-inositol incorporation followed by HPLC-coupled flow scintillation used to determine PtdIns(3)P, PtdIns(3,5)P₂ and PtdIns(4,5)P₂ levels from RAW cells exposed to vehicle alone, or 1 mM H₂O₂ 40 min (E), or 1 μM rotenone 60 min (F), in presence or absence of 20 nM apilimod. Data represent ± s.d. from three independent experiments. One-way ANOVA and Tukey’s post-hoc test used for E-F; * indicates statistical difference against control condition (P<0.05).