TRPML1 transcript levels determine PA effects on ROS elevation. (A) RT-qPCR (reverse transcription quantitative real-time PCR) analysis of siRNA-mediated TRPML1 knockdown (KD) efficiency in HUVECs (n = 3 repeats). Cells were transfected with a scramble (Scramble) or human TRPML1-targeting siRNA (ML1 KD). TRPML1 siRNA was co-transfected with Cy3-labeled siRNA (CY3) to monitor transfection efficiency. (B) Electrophysiological analysis of TRPML1 KD efficiency in HUVECs. Whole-endolysosome ITRPML1 was evoked by bath application of ML-SA5 (SA5, 3 μM). (C) The current densities of whole-endolysosome ITRPML1 (measured at −120 mV) in TRPML1 KD HUVECs (n = 5/4 endolysosomes for scramble/ML1 KD). (D) The effects of ML-SA5 (10 μM) treatment on PA-induced ROS elevation in TRPML1 KD HUVECs. Positive transfection of TRPML1 siRNA was confirmed with CY3 staining. (E) Quantitation of ML-SA5 effects on PA-induced ROS elevation in TRPML1 KD HUVECs. (F) RT-qPCR analysis of TRPML1 lentiviral overexpression in HUVECs (n = 3 repeats). (G) Representative traces of whole-endolysosome ITRPML1 evoked by ML-SA5 (SA5, 3 μM) in TRPML1-mScarlet-overexpressing HUVECs. Sham cells served as controls. (H) Current densities of whole-endolysosome ITRPML1 (measured at −120 mV) in TRPML1-overexpressing HUVECs (n = 7/4 endolysosomes for sham/ML1 OE). (I) The effects of PA on ROS levels in TRPML1-mScarlet-overexpressing HUVECs. (J) Summary of PA effects in TRPML1-mScarlet-overexpressing HUVECs, as shown in I. Average and individual results are shown. DCFDA intensities were measured and normalized to those in each control group. Scale bar = 10 μm. Data are presented as means ± SEMs; **P < 0.01, ***P < 0.001.