Journal: Genes & Development

Article Title: AMPK governs lineage specification through Tfeb-dependent regulation of lysosomes

doi: 10.1101/gad.274142.115

Figure Lengend Snippet: AMPK double-knockout EBs exhibit defects in lysosome function and Tfeb regulation. ( A ) GSEA plot showing enrichment of the gene set associated with the KEGG term “lysosome” in wild-type (WT) versus AMPK double-knockout (DKO) day 12 EBs in high glucose. ( B ) Relative mRNA levels of selected lysosomal genes in wild-type and AMPK double-knockout day 12 EBs in high glucose as assessed by qRT–PCR. n = 3 independent experiments. (*) P < 0.05; (#) P < 0.005 compared with wild type. ( C ) Plot of corrected log-transformed P -values of KEGG lysosome pathway enrichment in sets of genes identified to be up-regulated in wild type versus double knockout under high-glucose conditions. At each time point ( X -axis), the statistical significance of enrichment of genes associated with the KEGG term “lysosome” in wild-type versus AMPK double-knockout samples was determined as described in the Materials and Methods. The resulting P -values were log-transformed and are plotted on the Y -axis. Numbers in parentheses indicate the number of lysosomal genes significantly up-regulated in wild-type samples at each time point. ( D ) Direct fluorescence images of day 12 wild-type and AMPK double-knockout EBs after 1 h of incubation with DQ-BSA-Green followed by a 2-h chase prior to fixation. Bar, 100 µm. ( E ) Relative lysosomal activity, as determined by DQ-BSA assay, in day 12 wild-type and AMPK double-knockout EBs in high glucose. Results are from two independent experiments, with at least 10 EBs analyzed for each sample within an experiment. (****) P < 0.0001 compared with wild type . ( F ) Box and whisker plot of mRNA expression differences across 81 coordinated lysosomal expression and regulation (CLEAR) network genes in AMPK double-knockout versus wild-type samples at the indicated time points. High-glucose conditions only. See the Materials and Methods for details. (##) P < 10 −8 , one sample t -test for nonzero mean. ( G ) RT-qPCR analysis of Tfeb in day 8 wild-type and AMPK double-knockout EBs in high glucose. Three independent experiments were performed. (*) P < 0.05; (***) P < 0.0005 compared with wild type . ( H ) Immunoblot analysis of Tfeb in wild-type and AMPK double-knockout EBs differentiated in either high (HG) or low (LG) glucose for 8 d. ( I ) Time-course analysis of selected mTOR signaling components during high-glucose EB differentiation of wild-type and double-knockout ESCs. Lysates were immunoblotted with the indicated antibodies. ( J ) GSEA plot depicting enrichment of a “mTOR inhibition” gene set (derived from everolimus-treated vs. untreated mouse tissues) in wild-type versus double-knockout EBs at day 12. ( K ) Western blot analysis of day 6 wild-type and AMPK double-knockout EBs. After overnight incubation in the indicated glucose medium, EBs were treated with either vehicle (DMSO) or 500 nM INK128 for 2 h prior to lysate preparation and blotting with indicated antibodies. For standard bar graphs, average ± SEM is plotted. Statistical significance was determined by Student's t -test unless otherwise noted.

Article Snippet: Additional details can be provided on request. caTfeb was generated by amplifying a fragment of human Tfeb cDNA (Addgene, no. 44446) corresponding to the C-terminal 341 amino acids, adding a 5′ Kozak sequence and start codon. cDNA-pCIP constructs were linearized by ScaI digestion, repurified, and introduced into feeder-free ESCs using Lipofectamine 2000.

Techniques: Double Knockout, Quantitative RT-PCR, Transformation Assay, Fluorescence, Incubation, Activity Assay, Whisker Assay, Expressing, Western Blot, Inhibition, Derivative Assay

Journal: Genes & Development

Article Title: AMPK governs lineage specification through Tfeb-dependent regulation of lysosomes

doi: 10.1101/gad.274142.115

Figure Lengend Snippet: Proper lysosomal function is required for endoderm differentiation. ( A ) Immunoblot analysis of Tfeb in EBs derived from wild-type (WT) parental and two independent TfebMUT CRISPR clones. ( B ) Wild-type and TfebMUT ESCs underwent EB differentiation for 7 d in high (HG) or low (LG) glucose followed by RT-qPCR of selected CLEAR network genes. (*) P < 0.05; (#) P < 0.005 compared with wild-type LG. ( C ) Relative lysosomal activity, as determined by DQ-BSA assay, in wild-type and TfebMUT2 EBs. Data are from three independent experiments, with 10 EBs analyzed in each experiment. (****) P < 0.0001 compared with wild type . ( D , E ) RT-qPCR analysis of endoderm ( D ) and ectoderm ( E ) markers in day 7 EBs from wild-type and two TfebMUT clones. (*) P < 0.05; (#) P < 0.005 compared with wild type . ( F , G ) Wild-type ESCs were differentiated into EBs for 7 d in the presence or absence of 2.5 nM bafilomycin A (BafA). The compound was added daily. mRNA levels of selected endoderm ( F ) and ectoderm ( G ) genes were assessed by RT-qPCR. (*) P < 0.05; (#) P < 0.005 compared with DMSO. ( H ) IF analysis depicting wild-type EB-specific colocalization of GATA4, an endoderm marker, with the highly polarized staining of lysosomal marker Lamp2 in the outer layer of cells. Most EBs derived from AMPK double-knockout (DKO) ESCs lack appreciable staining throughout the structure, as shown in the bottom image. Bar, 50 µm. For B and D – G , data are from three independent experiments. For all bar graphs, average ± SEM is plotted. Statistical significance was determined by Student's t -test.

Article Snippet: Additional details can be provided on request. caTfeb was generated by amplifying a fragment of human Tfeb cDNA (Addgene, no. 44446) corresponding to the C-terminal 341 amino acids, adding a 5′ Kozak sequence and start codon. cDNA-pCIP constructs were linearized by ScaI digestion, repurified, and introduced into feeder-free ESCs using Lipofectamine 2000.

Techniques: Western Blot, Derivative Assay, CRISPR, Clone Assay, Quantitative RT-PCR, Activity Assay, Marker, Staining, Double Knockout

Journal: Genes & Development

Article Title: AMPK governs lineage specification through Tfeb-dependent regulation of lysosomes

doi: 10.1101/gad.274142.115

Figure Lengend Snippet: Tfeb overexpression corrects lysosomal defects and increases endodermal gene expression in AMPK double-knockout cells. ( A ) Schematic of wild-type (WT) Tfeb and caTfeb cDNA constructs. (TA) Transactivation domain; (bHLH) basic helix–loop–helix domain; (LZ) leucine zipper. S142 and S211 denote phosphorylation sites that control nuclear–cytoplasmic shuttling. ( B ) Western blots of representative GFP and caTfeb-expressing clones following stable transfection of cDNAs into an AMPK double-knockout (DKO) ESC CRISPR line. ( C ) RT-qPCR analysis of selected CLEAR target genes in wild-type parental and AMPK double-knockout clones expressing either GFP or caTfeb following 7 d of EB differentiation. ( D ) Direct fluorescence images of day 12 EBs derived from parental or caTfeb-expressing AMPK double-knockout ESCs after 1 h of incubation with DQ-BSA-Green followed by a 2-h chase prior to fixation. Bar, 100 µm. ( E ) Relative lysosomal activity, as determined by DQ-BSA assay, in day 12 wild-type and AMPK double-knockout (parental or caTfeb-expressing) EBs in high glucose. See the Materials and Methods for details. Results are from two independent experiments, with at least 10 EBs analyzed for each sample within an experiment. (****) P < 0.0001. ( F ,G) Wild-type parental and AMPK double-knockout ESCs expressing either GFP or caTfeb were differentiated for 7 d followed by RT-qPCR analysis of selected endoderm ( F ) and ectoderm ( G ) markers. C , F , and G show data from one representative experiment in which multiple GFP and caTfeb clones ( n = 3 per cDNA) were analyzed and compared with wild-type parental controls. Similar results were observed in four independent experiments with additional independently derived cDNA clones. (*) P < 0.05; (#) P < 0.005; (^) P < 0.0005. For all bar graphs, average ± SEM is plotted. Statistical significance was determined by Student's t -test.

Article Snippet: Additional details can be provided on request. caTfeb was generated by amplifying a fragment of human Tfeb cDNA (Addgene, no. 44446) corresponding to the C-terminal 341 amino acids, adding a 5′ Kozak sequence and start codon. cDNA-pCIP constructs were linearized by ScaI digestion, repurified, and introduced into feeder-free ESCs using Lipofectamine 2000.

Techniques: Over Expression, Gene Expression, Double Knockout, Construct, Phospho-proteomics, Control, Western Blot, Expressing, Clone Assay, Stable Transfection, CRISPR, Quantitative RT-PCR, Fluorescence, Derivative Assay, Incubation, Activity Assay

Journal: Genes & Development

Article Title: AMPK governs lineage specification through Tfeb-dependent regulation of lysosomes

doi: 10.1101/gad.274142.115

Figure Lengend Snippet: Model depicting how AMPK regulates cell fate decisions through lysosome-dependent control of Wnt signaling. ( A ) In wild-type cells, AMPK inhibition of mTOR allows sufficient levels of Tfeb to enter the nucleus and transcriptionally up-regulate endolysosomes. This organelle system is required for optimal signaling through the canonical Wnt pathway through its ability to sequester the GSK3β-containing DC, freeing β-catenin from degradation. Active β-catenin then translocates into the nucleus to induce target genes, many of which are important mediators of endodermal differentiation, including Sox17. ( B ) In AMPK double-knockout cells, increased mTOR signaling blunts Tfeb activity, leading to a defective endolysosomal compartment. As a result, DC inhibition of β-catenin remains intact even in the presence of Wnt, preventing robust activation of endodermal genes.

Article Snippet: Additional details can be provided on request. caTfeb was generated by amplifying a fragment of human Tfeb cDNA (Addgene, no. 44446) corresponding to the C-terminal 341 amino acids, adding a 5′ Kozak sequence and start codon. cDNA-pCIP constructs were linearized by ScaI digestion, repurified, and introduced into feeder-free ESCs using Lipofectamine 2000.

Techniques: Control, Inhibition, Double Knockout, Activity Assay, Activation Assay