Figure 4.

Trehalose affects MTOR activation leading to induction of TFEB nuclear translocation. (A, B) Macrophages were treated with or without Leucine and either trehalose (1 mM) provided 2 h before Leucine (labeled “pre”), trehalose (1 mM) provided at same time as Leucine (labeled “co”), or bafilomycin A₁ (200 nM) provided at same time a Leucine followed by: (A) Western blot analysis of MTORC1 downstream targets (phospho-Thr389 and total RPS6KB/S6K; phospho-Ser235/236 and total RPS6 ribosomal protein). Densitometric quantification from three separate experiments is shown to the right. (B) Immunofluorescence staining of MTOR colocalization with lysosome marker (LAMP2). Quantification of images is shown to the right graphed as colocalization percent of MTOR with LAMP2 (n ≥ 10 cells per group; scale bar: 5 μm). (C) Western blot analysis of TFEB in cytoplasmic and nuclear fractions of macrophages after treatment with 1 mM trehalose for 24 h ± MTOR inhibitor Torin. Densitometric quantification from n = 3 independent experiments is shown to the right. For all graphs are presented as Mean ± SEM. **P < 0.01 and ***P < 0.001, compare with vehicle; ^## P < 0.01 and ^### P < 0.001, compare with leucine only