Figure 2: Endogenous mitochondrion-generated ROS activate lysosomal TRPML1 channels and Ca²⁺ release.

(a) Upper panels: application of CCCP increased the fluorescence intensity of CM-H2DCFDA (green) versus the DMSO-treated control (CTL) group. The increase was inhibited by co-application of NAC (5 mM). Lower panels: representative traces of basal whole-endolysosomal currents under each condition (CTL, CCCP, CCCP+NAC) in TRPML1-expressing COS-1 cells. Scale bar, 50 μm. (b) Summary of CCCP pretreatment effects on basal I_TRPML1 and I_zTRPML1.1 from at least five patches for each experimental condition. Data are presented as mean±s.e.m. *P<0.05, ANOVA. Numbers of patches for each experimental condition are shown in brackets. (c) Effects of CCCP pretreatment on endogenous I_TRPML1 in HEK293 cells (mean±s.e.m., n=4–5 patches for each treatment).*P<0.05, ANOVA. (d) Pretreatment of CCCP (10 μM) for 1 h increased ML-SA1-induced Ca²⁺ release measured by Fura-2 imaging in TRPML1-expressing HEK293 cells. (e) CCCP pretreatment reduced GPN-induced lysosomal Ca²⁺ release, which is presumed to reflect the lysosomal Ca²⁺ store. Mean values (±s.e.m.) are shown for >30 cells per coverslip. (f) Quantification of results shown in d,e from at least three independent experiments (mean±s.e.m.). *P<0.05, paired t-tests.