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. 2016 May 12;12(8):1240–1258. doi: 10.1080/15548627.2016.1179405

Figure 2.

Figure 2.

Mechanistic analysis of TFE3 nuclear translocation induced by macrophage activation. (A) Immunoblots of TFE3-Ser321 phosphorylation state in nuclear and cytosolic fractions of RAW 264.7 cells incubated with DMSO (Ctrl.), LPS (24 h), or Torin-1 (2 h). (B) Representative western blots of RAW 264.7 cells treated with LPS for 1, 6, 24 and 48 h. Starvation with EBSS and treatment with Torin-1 were used as positive controls for MTORC1 inhibition. (C) Quantification of 2 independent experiments from (B). (D) MTOR and LAMP1 colocalization in RAW 264.7 macrophages treated with DMSO (Control) or LPS for 1, 6, 24 and 48 h. Starvation with EBSS serves as a positive control of the inactivation and dissociation of MTOR from lysosomal membranes. Insets are 2.6-fold magnification of the selected areas. Scale bars: 10 µm. (E) PPP3/calcineurin inhibition reduces TFE3 nuclear localization in RAW 264.7 macrophages treated with LPS. Pretreatment of cells with the PPP3/calcineurin inhibitor, FK506, reduces the number of cells with nuclear TFE3 after 24 h LPS treatment. * denotes P value < 0.05 by one-way ANOVA analysis (n = 3, > 470 cells per trial).