Fig. 5.

Characterization of candidate inhibitors on lysosomal Ca²⁺ stores in U2OS cells. A, representative traces of GPN induced Ca²⁺ release (top) and lysosomal disruption (bottom). U2OS cells were loaded with Fluo-4 AM and LysoTracker^® Red (LTR). Baseline fluorescence values were recorded for 15 min before addition of test compounds (30μM). Fluorescence was then monitored for 40 min before addition of GPN (300μM) to release lysosomal Ca²⁺. Measurements were made to quantify GPN evoked Ca²⁺ release (top) and loss of LTR fluorescence following initial drug addition (bottom) by assessing fluorescence ratio values in the regions highlighted by the green (peak F/F_o ratio) and red bars (minimal F/F_o ratio). PIC, protease inhibitor cocktail; BafA1, bafilomycin A1. B, Correlation plot depicting relationship between measurements of GPN induced calcium release (y-axis, green) and drug-induced lysosome disruption (x-axis red). Values are replotted from highlighted regions in (A). The cluster of non-lysomotropic compounds is highlighted (box). Data represents peak fluo-4 fluorescence ratios (F_fluo/F₀, where ‘F_fluo’ represents fluo-4 fluorescence at peak, and F₀ represents fluorescence at time = 0) and minimum LysoTracker^® fluorescence ratios (F_LTR/F₀, where ‘F_LTR’ represents minimum LysoTracker^® Red fluorescence prior to GPN addition, and F₀ represents fluorescence at time = 0). Compounds are labeled according to ranking # in Table 1.