Figure 2.
Small-molecule activation of lysosomal TRPML1 channels prevents PA-induced ROS elevation in human endothelial and epithelial cells. (A) Representative traces of basal (red), ML-SA5-activated (SA5, black), and ML-SI5-inhibited (SA5+SI5, blue) whole-endolysosome TRPML1-like currents (ITRPML1) in HUVECs. Currents were elicited with a ramp voltage protocol (−120 to +120 mV, 400-ms duration; only partial voltage ranges are shown) and a holding potential of 0 mV. (B) Dose-dependent effects of ML-SA5 and the more potent ML-SA8 on ITRPML1 (current amplitudes measured at −120 mV; n = 3 endolysosomes). ML-SA-induced currents were normalized to maximal responses with 1,000 nM SA5 and 300 nM SA8. (C) Dose-dependent effects of ML-SA5/8 co-treatment on PA (100 μM, 6 h)-induced ROS increases in HUVECs; BSA alone served as the control (CTL). (D) Summary of ML-SA5 effects on PA-induced ROS increases. For each experimental condition, ≥50 randomly selected cells from at least three independent experiments were analyzed. Relative ROS level was determined by normalizing DCFDA intensity to that of the CTL group. Mean (±SEM) and individual-cell data are shown. (E) Summary of ML-SA8 effects on PA-induced ROS increases. (F) Dose-dependent effects of ML-SA5 and ML-SA8 treatment on intracellular ROS levels. (G) Effects of ML-SA5 (10 μM) with and without ML-SI5 (30 μM) co-treatment on PA-induced ROS increases in HUVECs. (H) Quantitation of the results as shown in G. Average and individual results of ≥50 cells from three independent repeats are shown. (I) Effects of ML-SA5 (10 μM) or ML-SA8 (3 μM) with and without ML-SI5 (30 μM) co-treatment on PA-induced mitochondrial ROS (MitoSOX Green) increases in HUVECs. (J) Quantitation of data shown in I. (K) Effects of ML-SA5 (10 μM) with or without ML-SI5 (30 μM) co-treatment on PA-induced ROS increases in renal tubular epithelial cells (RTECs). (L) Quantitation of the data shown in K. Average and individual results from ≥50 cells are shown. Quantitative data are presented as means ± SEMs; ***P < 0.001.

Small-molecule activation of lysosomal TRPML1 channels prevents PA-induced ROS elevation in human endothelial and epithelial cells. (A) Representative traces of basal (red), ML-SA5-activated (SA5, black), and ML-SI5-inhibited (SA5+SI5, blue) whole-endolysosome TRPML1-like currents (ITRPML1) in HUVECs. Currents were elicited with a ramp voltage protocol (−120 to +120 mV, 400-ms duration; only partial voltage ranges are shown) and a holding potential of 0 mV. (B) Dose-dependent effects of ML-SA5 and the more potent ML-SA8 on ITRPML1 (current amplitudes measured at −120 mV; n = 3 endolysosomes). ML-SA-induced currents were normalized to maximal responses with 1,000 nM SA5 and 300 nM SA8. (C) Dose-dependent effects of ML-SA5/8 co-treatment on PA (100 μM, 6 h)-induced ROS increases in HUVECs; BSA alone served as the control (CTL). (D) Summary of ML-SA5 effects on PA-induced ROS increases. For each experimental condition, ≥50 randomly selected cells from at least three independent experiments were analyzed. Relative ROS level was determined by normalizing DCFDA intensity to that of the CTL group. Mean (±SEM) and individual-cell data are shown. (E) Summary of ML-SA8 effects on PA-induced ROS increases. (F) Dose-dependent effects of ML-SA5 and ML-SA8 treatment on intracellular ROS levels. (G) Effects of ML-SA5 (10 μM) with and without ML-SI5 (30 μM) co-treatment on PA-induced ROS increases in HUVECs. (H) Quantitation of the results as shown in G. Average and individual results of ≥50 cells from three independent repeats are shown. (I) Effects of ML-SA5 (10 μM) or ML-SA8 (3 μM) with and without ML-SI5 (30 μM) co-treatment on PA-induced mitochondrial ROS (MitoSOX Green) increases in HUVECs. (J) Quantitation of data shown in I. (K) Effects of ML-SA5 (10 μM) with or without ML-SI5 (30 μM) co-treatment on PA-induced ROS increases in renal tubular epithelial cells (RTECs). (L) Quantitation of the data shown in K. Average and individual results from ≥50 cells are shown. Quantitative data are presented as means ± SEMs; ***P < 0.001.

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