Fig. 4

The transcription factor EB (TFEB) does not mediate trehalose-induced upregulation of PGRN. a GRN mRNA levels were quantified from wild-type HeLa (WT) cells or HeLa cells stably over-expressing TFEB-GFP (TFEB-GFP) (n = 3 independent experiments run in duplicate). b WT or TFEB-GFP HeLa cells were fractionated into cytoplasmic/membrane and nuclear extracts and immunoblotted for total TFEB and PGRN. GAPDH and H3 were used to verify fractionation efficiency and were used as loading controls. Asterisk (*) denotes non-specific band in nuclear fraction. c Quantification of PGRN expression in WT or TFEB-GFP HeLa lysates in b. (n = 3 independent experiments). d TFEB-GFP HeLa cells were treated with trehalose (100 mM) or Torin1 (250 nM) for 0, 2, or 24 h and imaged live with a fluorescent microscope to visualize TFEB-GFP localization. e TFEB-GFP HeLa cells were treated with vehicle, trehalose (100 mM), or Torin1 (250 nM) for 24 h and then fractionated into cytoplasmic/membrane and nuclear extracts and immunoblotted for total TFEB and PGRN. Asterisk (*) denotes non-specific band in nuclear fraction. f Quantification of nuclear TFEB (top) and cytoplasmic/membrane PGRN (bottom) from the immunoblots in e. (n = 3 independent experiments). g Immunoblots from whole-cell lysates of HAP1 WT and TFEB KO cells showing absence of TFEB expression. h GRN mRNA levels were quantified from HAP1 wild-type (WT) and TFEB knock-out (TFEB KO) cell lines after treatment with vehicle or trehalose (100 mM) for 18 h (n = 3 independent experiments run in duplicate). i HAP1 WT and TFEB KO cells were treated with vehicle or trehalose (100 mM) for 24 h and then fractionated into cytoplasmic/membrane and nuclear extracts. Immunoblots for PGRN (cytoplasmic/membrane) and TFEB (nuclear) are shown. j Quantification of PGRN (top) and TFEB (bottom) from the immunoblots in i (n = 3 independent experiments). In all graphs, the bars represent the mean ± SEM. For a, c; *differs from WT, P < 0.05, **P < 0.01, using unpaired two-tailed Student’s t-test. For f, h, j; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 using one-way ANOVA followed by Tukey’s comparison post-hoc test