Fig 2: NEMEP interacts with GLUT1/GLUT3 and facilitates glucose uptake.Physical interactions of mouse (a) human (b) NEMEP with GLUT1 and GLUT3. Lysates from HEK293T cells co-transfected with plasmids encoding GLUT1-FLAG or GLUT3-FLAG or control vector and NEMEP-HA (as indicated) were immunoprecipitated with anti-FLAG affinity beads, and immune complexes were analyzed by immunoblotting using an antibody against HA. The protein inputs were detected with western blotting using indicated antibodies. c Immunoprecipitation of endogenous NEMEP from NEMEP-3XFLAG knock-in mESCs using anti-FLAG antibody. The immune complexes were analyzed by immunoblotting using antibodies against FLAG and GLUT1 and GLUT3. The protein inputs were detected with western blotting using indicated antibodies. d Lysates from HEK293T cells co-transfected with plasmids encoding mGLUT1-FLAG or mGLUT3-FLAG and NEMEP (GFP-tagged, wild type or indicated mutant variants) immunoprecipitated with anti-GFP-trap affinity beads; immune complexes were analyzed by immunoblotting using an antibody against FLAG. The protein inputs were detected with western blotting using indicated antibodies. Glucose consumption (e) and lactate excretion (f) in WT and CRISPR-dCas9-VP64 (CRISPRa) mediated NEMEP overexpressing mESCs. Data are means ± S.E.M., n = 3 biological independent samples. g WT and CRISPR-dCas9-VP64 (CRISPRa) mediated NEMEP overexpressing mESCs were supplied with 25 mM glucose, 2 µM of oligomycin (ATP synthase inhibitor), and 50 mM 2-DG (a glucose analog that inhibit glycolysis) at the indicated time. ECAR was examined using a Seahorse XFe96 analyzer. The values are normalized to the protein concentration (means ± S.E.M., n = 8 biological independent samples). h Glucose uptake analysis in mESCs cells stably expressing control plasmid or plasmids with WT or mutant variants of NEMEP, as indicated. The values are normalized to the protein concentration (means ± S.E.M., n = 3 biological independent samples). i,j Glucose uptake analysis in HEK293T cells expressing plasmids for overexpression of the indicated proteins or protein pairs. The values are normalized to the protein concentration (means ± S.E.M., n = 3 biological independent samples). a–j: Data are the representative of three independent experiments with similar results. P values were determined by unpaired two-tailed t-test (e, f), one-way ANOVA (h) with Sidak’s corrections or two-way ANOVA (i, j) with Tukey’s corrections. Source data are provided as a Source Data file.

Fig 3: Nodal signaling mediates glucose uptake during mesendoderm differentiation.a Glucose uptake analysis in control and Activin A (AC)-treated for 2 h EBs. The values are normalized to the protein concentration (n = 4 biological independent samples). b Glucose uptake analysis in Activin A (AC)-treated for 2 h or untreated WT and Cripto KD/Cryptic KD EBs. The values are normalized to the protein concentration (n = 3 biological independent samples). c Glucose uptake analysis in Activin A (AC)-treated for 2 h or untreated WT and Smad2 KO/Smad3 KD EBs. The values are normalized to the protein concentration (n = 3 biological independent samples). d Glucose uptake analysis in Activin A (AC- treated for 2 h or untreated WT and Trim33 KO EBs). The values are normalized to the protein concentration (n = 3 biological independent samples). e Glucose uptake analysis of Activin A (AC)-treated for 2 h or untreated WT and Nemep KO EBs at day 3. The values are normalized to the protein concentration (n = 3 biological independent samples). f Glucose uptake analysis of NEMEP-FLAG overexpressing WT and Nemep KO EBs at day 3. The values are normalized to the protein concentration (n = 3 biological independent samples). g Working model for the role of NEMEP during mesendoderm differentiation (Created with BioRender.com). A highly conserved putative lncRNA Gm11549 is specifically induced by Nodal signaling during mesendoderm differentiation and it is essential for mesendoderm differentiation in mESCs. There is a “ORF” hidden in Gm11549 that codes a transmembrane micropeptide, we named NEMEP. NEMEP functions by interacting with GLUT1 and GLUT3, and augment the glucose uptake capacity of glucose transporter proteins to meet the energy needs during mesendoderm differentiation. a–f: Data are the mean ± S.E.M. P values were determined by unpaired two-tailed t-tests (a), one-way ANOVA (e, f) with Dunnett’s corrections, two-way ANOVA (b–d) with Sidak’s corrections, and data are representative of three independent experiments with similar results. Source data are provided as a Source Data file.