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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. 7 — (a) Experimental plan for Seahorse mitochondrial stress tests shown through c-h. (b) Representative IB for FLAG in NIH3T3 cells expressing the indicated FLAG-tagged NDRG1 wild type (WT) or FLAG-tagged Ser/Thr>Ala mutant of NDRG1 (n = 3 independent experiments). (c) Seahorse mitochondrial stress test in serum-deprived NIH3T3 cells expressing FLAG-tagged BNIP3 WT or FLAG-tagged Ser79Ala or Ser88Ala mutant BNIP3 in presence of 0.25 mM OA, followed by sequential addition of oligomycin (O), FCCP (F), and rotenone + antimycin (R + A) to assess mitochondrial respiratory function (n = 5 independent experiments). (d-f) Seahorse mitochondrial stress tests in NIH3T3 cells expressing the indicated FLAG-tagged NDRG1 WT or Ser/Thr>Ala or Ser/Thr>Asp mutant of NDRG1. Quantifications for mitochondrial respiratory function in cells expressing (g) FLAG-tagged NDRG1 WT or Ser/Thr>Ala mutants of NDRG1, or (h) FLAG-tagged NDRG1 WT or Ser336Ala and Ser336Asp mutants of NDRG1 are shown. For d-h (n = 5 independent experiments). (i) Cartoon depicting experimental plan for confocal microscopy performed in j and k. Representative blots for NDRG1 in siCon and siNdrg1 NIH3T3 cells are shown in i, right (n = 5 independent experiments). (j) MitoTracker CMXRos red fluorescence in siCon or siNdrg1 cells (siCon 58 cells and siNdrg1 62 cells from n = 3 independent experiments). (k) MitoTracker CMXRos red fluorescence in FLAG (green fluorescence)-tagged WT or phosphorylation-deficient mutants of NDRG1 (NDRG1^WT 37 cells, NDRG1^Thr328Ala 34 cells, NDRG1^Ser332Ala 32 cells, and NDRG1^Ser336Ala 33 cells from n = 4 independent experiments). Ponceau is loading control. For c-f, values are mean ± SEM. For g, h, j and k, individual replicates and means are shown. *P < 0.05, **P < 0.01 and ***P < 0.001, One-way ANOVA followed by Tukey’s multiple comparisons test (g, h and k); two-tailed unpaired Student’s t-test (j). 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. 7.

Source data