Figure 7. The clearance of SK1-I-induced vacuoles occurs concomitantly with the activation of TFEB-mediated lysosome biogenesis and is dependent on Atg5.

(A) Live cell fluorescence images of WT and Atg5−/− MEFs stably expressing GFP-TFEB treated with 7.5 μM SK1-I for 16 h. White and yellow dotted lines represent outlines of nuclei lacking or containing GFP-TFEB, respectively.

(B) WT or Atg5−/− MEFs were treated with 10 μM SK1-I for 24 h and fractionated to obtain cytoplasmic (Cyt), nuclear binding proteins (NBP) or insoluble nuclear proteins (INB). Immunoblot for TFEB, α-tubulin and PARP.

(C) WT MEFs were treated with DMSO or 10 μM SK1-I for the indicated time points. Cell lysates were analyzed by immunoblot for Lamp1, Rab7 and β-actin.

(D) WT and Atg5−/− MEFs were pulsed with 100 μg/mL AF488-Dex for 1 h followed by a 2.5 h chase in dextran-free DMEM to label lysosomes. Live cell fluorescent images of cells treated with DMSO or 10 μM SK1-I for 4 h followed by MagicRed Cathepsin B substrate (MR-CathB).

Scale bars represent: 20 μm in (A); 10 μm in (D).