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. 2021 Mar 22;28(9):2555–2570. doi: 10.1038/s41418-021-00770-7

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a Wnt3a-mediated nuclear-localized TFEB does not induce lysosomal biogenesis. TFEB-EFGP stable cells were treated with the indicated media for 4 h. Acidic organelles were visualized by treatment with the LysoTracker dye. Both Wnt3a-CM treatment and glucose starvation induced nuclear localization of TFEB-EGFP (Bottom). Only glucose starvation, but not Wnt3a-CM treatment, induced the increased number of lysosomes (Top). Quantification of number of acidic organelles/cells was shown in the right panel. Scale bar, 10 μm. b Heat map analysis of expression profiles after treatment with Wnt3a-CM under siGFP or siTFEB conditions. About 27% genes among the genes upregulated by Wnt3a-CM treatment was TFEB-dependent. c, d Top 10 GO analysis of “TFEB-mediated Wnt target genes”. Bar chart shows classification of Biological Processes (c) or Molecular Function (d). Bars represent the p value for the specified category. e Confirmation of “TFEB-mediated Wnt target genes”. siGFP or siTFEB transfected HeLa cells were treated with L-CM or Wnt3a-CM for 6 h and qPCR was performed to measure expression of randomly selected genes among “TFEB-mediated Wnt target genes”. f, g Wnt signaling did not increase the expression of “TFEB-mediated Wnt target genes” upon TNKS1 (f) or TNKS1/2 (g) knockdown as well as TFEB knockdown. siGFP, siTFEB, siTNKS1, or siTNKS1/2 transfected HeLa cells were treated with L-CM or Wnt3a-CM. qPCR was performed to measure expression of “TFEB-mediated Wnt target genes”. h Co-transfection TFEB and TNKS1 increased expression of “TFEB-mediated Wnt target genes”. qPCR was performed for “TFEB-mediated Wnt target genes” in TFEB-EGFP and Flag-TNKS1 expressing HeLa cells. Data information: In (e–g), data are presented as mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.005 (Student’s t test).