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. 2021 Jan 20;12:476. doi: 10.1038/s41467-020-20780-2

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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. 3 — a, b Representative western blots and quantitative results of p-mTOR and mTOR’s substrates (p-ULK1, p-p70S6K, and p-4EBP1) in wild-type and ENDOG knockout L02 cells following the starvation for 6 h (n = 4 independent samples, data are presented as mean values ± SD, *p < 0.05; **p < 0.01; ***p < 0.001). c, d Representative western blots and quantitative results of the indicated mTOR signaling pathway proteins in Endog^+/− and Endog^−/− mouse liver after starvation for 24 h (n = 3 independent animals, data are presented as mean values ± SD, *p < 0.05; **p < 0.01; ***p < 0.001). e, f Representative western blots and quantitative results of the indicated mTOR signaling pathway proteins in ENDOG and RHEB/RHEB^Q64L co-expressed L02 cells (S: short time exposure; L: long time exposure; n = 3 independent experiments, data are presented as mean values ± SD, *p < 0.05; **p < 0.01; ***p < 0.001). g, h Representative immunofluorescence images of GFP-LC3 puncta (g) and respective quantitative results (h) in ENDOG and RHEB^Q64L co-overexpressed L02 cell under normal and HBSS + CQ conditions (WT: wild-type; OE: ENDOG overexpressed; RHEB^Q64L transiently transfected for 48 h; starvation for 6 h; CQ: 50 μM for 6 h; scale bar = 10 μm; n = 100 independent cells examined over 3 independent experiments; data are presented as mean values ± SD, *p < 0.05; ***p < 0.001). i Co-IP experiments showed that ENDOG overexpression decreases interactions between 14-3-3γ with TSC2/Vps34 (L02 ENDOG-KO cell co-overexpressed Myc-14-3-3γ with Flag-ENDOG or empty vector for 48 h; biological repeated three times). j Endogenous Co-IP experiments showed that starvation enhances the interaction between ENDOG and 14-3-3γ but weaken the interaction of TSC2/Vps34 with 14-3-3γ (in L02 WT cell; Star.: starvation for 12 h; S: short time exposure; L: long time exposure; biological repeated three times). k, l Representative western blots and quantitative results of the mTOR pathway and autophagy-related proteins in ENDOG or 14-3-3γ overexpressing L02 cells (ENDOG or 14-3-3γ transiently transfected for 48 h; n = 3 independent experiments; data are presented as mean values ± SD, *p < 0.05; **p < 0.01, ***p < 0.001). m Representative immunofluorescence images of GFP-LC3 puncta (upper) and respective quantitative result in ENDOG or 14-3-3γ overexpressing L02 cells (lower). (HBSS: starvation for 6 hours; CQ, 50 μM for 6 h; scale bar = 10 μm; n = 100 independent cells examined over 3 independent experiments; data are presented as mean values ± SD,; ***p < 0.001). Source data are provided as a Source data file.