Fig 4. ROS accelerate recovery of lysosome size and number upon PIKfyve reactivation.

(A) Top two rows: RAW cells pre-labelled with Lucifer yellow were exposed to either vehicle, 1 mM H₂O₂ 40 min, 1 μM rotenone 60 min, 10 μM CDNB 30 min, or 5 μM MCB 30 min. Bottom two rows: alternatively, RAW cells were first treated with 20 nM apilimod for 60 min (0 h), followed by apilimod removal and replenishment with complete media for 2 h in the presence of vehicle, H₂O₂, rotenone, CDNB, or MCB at previously indicated concentrations. 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). Data are represented 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, where * indicates statistically significant difference between control conditions (P<0.05).