Figure 5: TRPML1 channel activity is required for ROS-induced TFEB-nuclear translocation.
From: MCOLN1 is a ROS sensor in lysosomes that regulates autophagy
(a) Differential effects of BAPTA-AM and NAC on CCCP- and Torin-1-induced TFEB-nuclear translocation in HEK293 cells stably expressing mCherry–TFEB. Cells were treated with CCCP (5 μM) and Torin 1 (1 μM) for 1 h to induce TFEB-nuclear translocation. Nuclei were counterstained with DAPI (pseudo-colored in green). Scale bar, 10 μm. (b) Ratio of nuclear versus cytosolic TFEB (>100 cells per experimental condition). (c) Western blot analysis of cytosolic versus nuclear pools of TFEB proteins with and without CCCP treatment, and in the presence and absence of NAC and BAPTA-AM. Tubulin and Lamin are proteins abundant in the cytosolic and nuclear fractions, respectively. (d) Averaged effects of NAC or BAPTA-AM on CCCP-induced TFEB-nuclear translocation, based on multiple repeated experiments as shown in c. (e) Differential effects of ML-SI3 on CCCP- and Torin-1-induced TFEB-nuclear translocation, shown with western blot analyses of TFEB. (f) The quantitative effects of ML-SI3 on CCCP-induced TFEB-nuclear translocation based on the multiple repeated experiments as shown in e. (g) CCCP (10 μM for 1 h) induced accumulation of TFEB, detected by anti-human TFEB antibody, in the nuclei of WT, but not ML-IV cells. In contrast, Torin-1 induced TFEB-nuclear translocation in both WT and ML-IV cells. Nuclei were labelled with DAPI (pseudo-colored in red). Scale bar, 10 μm. (h) Average ratios of nuclear versus cytosolic TFEB immuoreactivity (>100 randomly-selected cells per experiment). ML-SI4 (10 μM) inhibited TFEB-nuclear translocation induced by CCCP (10 μM) treatment for 1 h. (i) The effects of ML-SA5 (1 μM for 1 h) on TFEB-nuclear translocation in the presence and absence of ML-SI4 (10 μM) in WT and ML-IV cells. Scale bar, 10 μm. (j) Average ratios of nuclear versus cytosolic TFEB immuoreactivity (>50 randomly-selected cells per experiment). All quantification data are presented as mean±s.e.m.; *P<0.05, paired t-test for western blots and ANOVA for all other comparisons.
