Fig. 3: Co-activation of endogenous TPC2 evokes global Ca²⁺ signals.

a, b Effect of TPC2-A1-N (N; 30 µM), TPC2-A1-P (P; 60 µM) or a combination of the two (N+P). on Ca²⁺ levels of individual naïve (untransfected) HeLa cells loaded with Fura-2. Each trace is the fluorescence ratio response of a single cell imaged from a typical field of view (a). The thicker trace is the average of the population. External Ca²⁺ was removed (0 Ca) prior to stimulation. Pooled data (mean ± sem) quantifying the peak change in ratio from 12 to 15 experiments where each point represents the mean response of all cells from an independent experiment, are shown in b. *p = 0.02, **p = 0.004 (Kruskal-Wallis test followed by Dunn’s post hoc test). c, d Effect of the agonist combination on Ca²⁺ levels where the agonists were added simultaneously (N+P) or when TPC2-A1-N was added after TPC2-A1-P (P > N) c. Data are mean ± sem from 3 to 4 independent experiments. Pooled data quantifying the peak change in ratio is shown in d. *p = 0.03, **p = 0.004 (One-way ANOVA followed by Dunnett’s post hoc test). e Structures of the inactive TPC2-A1-N analogue, SGA-10 and the inactive TPC2-A1-P analogue, SGA-153. f, g Effect of SGA-10 (10; 30 µM) and SGA-153 (153, 60 µM) on Ca²⁺ levels. Cells were co-stimulated with TPC2-A1-N or TPC2-A1-P as indicated. Pooled data (mean ± sem from 3 to 8 experiments) quantifying the peak change in ratio is shown in g. *p = 0.04, ****p < 0.0001 (One-way ANOVA followed by Dunnett’s post hoc test). h–m Effect of TPC2^L265P-GFP or LAMP1-GFP on Ca²⁺ responses to TPC2-A1-N (30 µM) and TPC2-A1-P (60 µM). Cells were transiently transfected and segregated according to whether they were GFP-positive or -negative. Results are shown as responses of individual cells from a typical field of view (h, k) or as mean ± sem from 3 experiments (i, l). Pooled data quantifying the peak change in ratio are shown in j and m. Epifluorescence images of GFP and Fura-2 (380 nm excitation) from a typical field of view showing transfected (+) and non-transfected (–) cells. **p = 0.008, n.s. not significant (Unpaired t-test, two-tailed). n CRISPR targeting strategy for knockout of TPC2 in SK-MEL-5 cells (top) and qPCR validation (bottom) presented as mean ± sem from 3 experiments. ****p < 0.0001 (Unpaired t-test, two-tailed). o, p Effect of TPC2-A1-N (10 µM or 30 µM) and/or TPC2-A1-P (30 µM or 60 µM) on cytosolic Ca²⁺ in wild-type (WT) and TPC2 knockout (KO) SK-MEL-5 cells (o). Dare are mean ± s.e.m from 3 to 19 experiments. Pooled data quantifying the peak change in ratio in response to the indicated agonist concentration are shown in p. *p = 0.01, **p = 0.005, n.s. not significant (Unpaired t-test, two-tailed); ****p < 0.0001 (Mann-Whitney test, two-tailed). Source data are provided as a Source Data file.