Figure 2.

HEPES promotes MiT/TFE nuclear translocation independent of MTORC1 activity. (A) Representative images and (B) quantified MiT/TFE nuclear import in RAW cells treated with HEPES (H), sucrose (S), or Torin1 (T) for 6 h, stained for endogenous TFEB, TFE3, or MITF levels (in green) and counterstained with DAPI (in blue). Values are expressed as percent of cells counted (>100 per experiment). (C) Western blot analysis on cytosolic and nuclear fractions isolated from RAW macrophages treated for 6 h, as indicated. Membranes were probed with antibodies against MiT/TFE family members. TUBA and LMNB1 were used as controls for the cytosolic and nuclear fractions, respectively. (D) HEPES and sucrose supplementation to RAW cell culture media does not inhibit MTORC1 signaling. Western blot analysis on protein extracts isolated from RAW cells treated for 6 h as indicated in the presence and absence of the PtdIns3K inhibitor LY294002 (LY2; 50 µM). Membranes were probed with antibodies against p-EIF4EBP1 (Thr37/46), p-RPS6 (Ser235/236), and TFEB. (E-G) LY2 prevents HEPES-dependent MiT/TFE nuclear redistribution and lysosome biogenesis. (E) Representative images and (F) western blot analysis of MiT/TFE relocalization in RAW cells pretreated with LY2 for 30 min and subsequently cultured in either DMEM or DMEM^+H for 6 h. (G) Flow cytometric analysis of LTG-stained cells pre-treated with LY2 and grown in DMEM or DMEM^+H for another 16 h. (H-I) HEPES perturbs lysosomal pH/acidification. (H) Flow cytometric analysis of fluorescent intensity (FL1) in LysoSensor™ DND-189 stained RAW cells treated for 2 h, as indicated. (I) Quantified lysosomal pH using LysoSensor™ Yellow/Blue DND-160 in cells grown in DMEM or DMEM^+H for 4 h. (J) Flow cytometric analysis of LTG-stained RAW cells grown in DMEM supplemented with HEPES (20 mM), MES (20 mM), PIPES (10 mM), sucrose (80 mM), and Tris-HCl (20 mM) for 16 h. Values are expressed as mean ± SEM, n = 3-4 in A-J. *P < 0.05, **P < 0.01.