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. 2023 Jun 29;25(7):989–1003. doi: 10.1038/s41556-023-01163-3

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© The Author(s) 2023

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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Extended Data Fig. 8 — (a) Representative confocal images of NIH3T3 cells silenced for Sgk1/2/3, Ndrg1 or Akt1/2 and cultured in serum-free medium for 30 min in presence of MitoTracker green. Magnified insets are shown. Quantifications for mitochondrial number and mitochondrial size/shape descriptors are shown (siCon 71 cells, siSgk1/2/3 36 cells, siNdrg1 62 cells, and siAkt1/2 36 cells from n = 5 (siCon) and n = 3 (siSgk1/2/3, siNdrg1, and siAkt1/2) independent experiments). (b) IB and quantifications for indicated proteins in livers of 3–4 mo-old male mice injected with siRNAs against Sgk1, Akt1/2 or Ndrg1 and subjected to 14–16 h fasting. N values for number of mice analyzed for indicated proteins are in parentheses. SGK1: siCon (n = 4) and siSgk1 (n = 3); AKT1 and 2: siCon (n = 4) and siAkt1/2 (n = 4); NDRG1: siCon (n = 4) and siNdrg1 (n = 3); P-NDRG1^Thr346/NDRG1: siCon (n = 4), siSgk1 (n = 4) and siAkt1/2 (n = 3). (c) AUC for OCR in livers of 14–16 h fasted Con (n = 5), siSgk1 (n = 3), siNdrg1 (n = 4), and siAkt1/2 (n = 3) mice. (d) Immunohistochemistry and quantification for equivalent FLAG expression in livers silenced for endogenous Ndrg1 and then injected with FLAG-tagged WT or Ser336Ala NDRG1 plasmid (n = 3 mice). 2° Ab-only control is shown. (e) Cartoon depicting experimental plan performed in Fig. 5a and Extended Data Fig. 8f. (f) Representative live-cell imaging of mCherry-NDRG1^WT or mCherry-NDRG1^Ser336Ala and ER-Tracker green in NIH3T3 cells cultured in serum-free medium for 30 min. Magnified insets are shown. White arrowheads: NDRG1 (mCherry)/ER (ER-Tracker) contacts. (h) Quantification for % colocalization of mCherry with ER-tracker in g. Values are mean ± SEM (n = 3 independent experiments). Please refer to Supplementary Video 7 (mCherry-NDRG1^WT/ER-Tracker), and Supplementary Video 8 (mCherry-NDRG1^Ser336Ala/ER-Tracker). Ponceau is loading control. Individual replicates and means are shown. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, One-way ANOVA followed by Tukey’s multiple comparisons test (a, c and d); two-tailed unpaired Student’s t-test (b); 2-way ANOVA followed by Tukey’s multiple comparisons test (g). ns=not significant. Please refer to Supplementary Table 10_statistical summary. Source numerical data are available in SourceData_Table 1, and unprocessed blots are available in Source Data Extended Data Fig. 8.

Source data