Figure 6.

TFEB activation is necessary but not sufficient for upregulation of lysosomal function following mTORC1 suppression. (A) MEFs were first transiently transfected with a TFEB luciferase reporter vector together with Renilla luciferase vector for 48 h. Cells were then treated with EBSS or PP242 (1 μM) for 4 h. Data were presented as mean ± SD from two independent experiments (each in triplicates) (^**P< 0.01 comparing to the control, Student's t test). (B) MEFs were first transiently transfected with the Flag-tagged TFEB for 48 h, and then treated with EBSS, rapamycin (1 μM) or PP242 (1 μM) for 2 h. At the end of treatment, cell lysate was collected and subject to immunoblotting. (C) MEFs were treated with EBSS, rapamycin (1 μM) or PP242 (1 μM) for 4 h, and the mRNA levels were measured by RT-qPCR. Values are expressed as fold increase compared to the control group. Data are presented as mean ± SD from two independent experiments (each in triplicates) (^*P< 0.05, ^**P < 0.01 comparing to their respective group in the control cells, Student's t test). (D) TFEB mRNA levels measured by qPCR. Data were presented as mean ± SD from two independent experiments (each with duplicates) (^**P< 0.01, Student's t test). (E) After transfection with TFEB siRNA or scrambled siRNA for 48 h, cells were treated with EBSS or PP242 (1 μM) for 3 h, then cathepsin B and L activities were determined as in Figure 2B. (F) Atg5-WT and Atg5-KO MEFs were transfected with TFEB luciferase reporter vector together with Renilla luciferase vector for 48 h, cells were then treated in EBSS for 4 h. The luciferase activity was determined and presented as in panel (A). (G) Atg5-WT and Atg5-KO MEFs were first transiently transfected with the Flag-tagged TFEB for 48 h, and then treated with EBSS for 2 h. Cell lysates were collected and subjected to cytoplasm and nucleus subfraction, as described in panel (B).