TFEB-family transcriptional factors that regulate lysosome biogenesis mediate the antioxidant and mitophagy-stimulating effects of TRPML1. (A) The oxidant Ch-T activated whole-endolysosome ITRPML1 in HUVECs. (B and C) Representative traces of ML-SA5 (3 μM)-induced whole-endolysosome ITRPML1 in BSA-treated (B) and PA-treated (C) HUVECs. (D) Quantitation of whole-endolysosome ITRPML1 in HUVECs with or without PA treatment. ML-SA5-induced currents at −120 mV in B (CTL) and C (PA) were normalized to the endolysosome size/capacitance (pF, n = 4 endolysosomes). (E) Effects of ML-SA5 (10 μM) on PA-induced ROS elevation in wild-type (WT) and MITF/TFEB/TFE3 triple knockout (TKO) HeLa cells. (F) Western blot analysis of MITF/TFEB/TFE3 expression in WT and MITF/TFEB/TFE3 TKO Hela cells. β-Actin served as the loading control. (G) Quantitation of ML-SA5 effects as shown in E. (H) Effects of ML-SA5 treatment on subcellular TFEB localization in PA-treated HUVECs, in which TFEB-mScarlet was stably expressed. (I) Quantitation of data in H based on ratios of TFEB-scarlet nuclear/cytoplasmic intensity; ≥50 randomly selected individual cells from three experimental repeats were analyzed. (J) Western blot analysis of ML-SA5 effects on the phosphorylation status of TFEB in TFEB-mScarlet stable HUVECs. GAPDH served as the loading control. (K) Quantitation analysis of data in J. The ratios of pS211 versus total TFEB were measured and normalized to that in the control group (n = 3 repeats). (L) Effects of ML-SA5 on Parkin puncta number in PA-treated Parkin-mScarlet stable HUVECs. (M) Quantitative analysis of data in L; ≥50 randomly selected cells from three experimental repeats were analyzed. Data are presented as means ± SEMs; **P < 0.01, ***P < 0.001. Source data are available for this figure: SourceData F5.