(A) Inhibition of PIKfyve using two different concentrations of Apilimod (25 nM and 250 nM) led to a strong decrease in the number of acidified organelles labelled with lysotracker Red DND-99. (B) Quantification of the average cellular number of lysotracker positive vesicles measured using Squassh analysis in the MOSAIC suite in ImageJ. Both 25 nM and 250 nM Apilimod significantly reduced the number of lysotracker positive vesicles. (C) Quantification of average vesicular lysotracker intensity showed a small but significant reduction in vesicular lysotracker staining with 25 nM Apilimod and a stronger reduction with 250 nM. (A–C) Data were pooled from three independent experiments, yielding a total of ≥309 cells per condition. Error bars are S.E.M. (D) Analysis of the LampI positive compartment upon inhibition of the PIKfyve complex using 25 nM and 250 nM Apilimod. Both concentrations of Apilimod reduced the number of LampI positive vesicles. (E) Quantification of the average number of LampI positive vesicles per cell and its dependence on PIKfyve showed that its inhibition reduced the number of LampI positive late endosomes and lysosomes. (F) Analysis of average size of LampI positive late endosomes/lysosomes showed that although the number of LampI vesicles per cell was reduced, their size increased, suggesting swelling or aggregation of the compartment. Data presented in (E) and (F) were pooled from three independent experiments with 240 cells analysed per condition. Error bars are S.E.M. Statistical analysis in (B), (C), (E) and (F) was performed using one-way ANOVA with Tukey's post-hoc test, α=0.05, ***P≤0.001, ****P≤0.0001.