Extended Data Fig. 10: Cellular events associated with PA-induced TFEB activation.
From: TFEB–GDF15 axis protects against obesity and insulin resistance as a lysosomal stress response
a, Effect of ML-SI3, a TRPML1 inhibitor, on PA-induced TFEB nuclear translocation (right). Representative immunofluorescence pictures are presented (left). Scale bars, 5 μm. b, Mitochondrial ROS stained with MitoSOX in peritoneal macrophages treated with BSA or PA (400 μM) for 1 h in the presence or absence of Mito-Tempo (500 μM) pretreatment (lower). Gating strategy and representative scattergrams are presented (upper and middle). c, Macrophages were treated with PA or BSA control, and cell extract was subjected to immunoblot analysis to study phosphorylation of mTOR and its target, 4EBP1. Representative immunoblot (left). Densitometric ratio of phospho-mTOR or -4EBP1 band to HSP90 band (right). d, Macrophages were treated with PA or BSA control were subjected to immunofluorescence, and colocalization between mTOR and LAMP1 was examined (right). Representative immunofluorescence pictures are presented (left). Scale bars, 5 μm. e, Raw264.7 cells transfected with pcDNA 3.1-HA or HA-DCnA-H151Q were treatment with 400 μM PA or BSA. After immunostaining with anti-HA and TFEB antibodies, cells were analyzed by confocal microscopy (yellow arrow head, HA-DCnA-H151Q-transfected cells showing no TFEB translocation after PA treatment; red arrow head, untransfected cells showing TFEB translocation after PA treatment). Scale bars, 5 μm. All data are shown as means ± SEM. ***P < 0.001 by one-way ANOVA with Tukey’s test. (n.s., not significant).
