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. 2022 Jul 21;4(7):856–866. doi: 10.1038/s42255-022-00605-w

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© The Author(s) 2022

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. 1 — a, Quantification of nuclear TFEB levels from BMDMs treated or not for 30 min with heat-killed Mycobacterium tuberculosis (hk Mt, 10 µg/mL), living Salmonella Typhimurium (SmT, MOI 5), or LPS/IFNγ (15 min), related to Fig. 1a-c. Quantification represents (a) N = 101, 117 (b) N = 56, 115, (c) N = 109, 99 cells examined over n = 2 independent experiments. Red lines indicate the mean and p-values were determined using unpaired, two-sided Student’s t-test. b, Quantification of cellular and nuclear TFEB induction in the TFEB activation mimic and GFP-expressing control cells, quantified from images of cells immune-stained against endogenous TFEB. Scale bar: 10 µm. Box plots (box: 25–75 percentile, middle line: median, whiskers: 5-95 percentile) of N = 52 (GFP), N = 41 (TFEB) cells examined over of n = 2 independent experiments. P values were calculated using unpaired, two-sided Student’s t-test. c, Heatmaps of lysosomal genes from RNA-seq analysis in: (left panel) naïve versus 24 h LPS/IFNγ treated, GFP-expressing BMDMs; (right panel) naïve BMDMs expressing TFEB-GFP or GFP. d, Quantification of LysoTracker fluorescence in TFEB-GFP- and GFP-expressing BMDMs by flow cytometry. Bars represent mean ± s.d. of n = 3 independent experiments. Pvalues were calculated using two-tailed one-sample t-test. MFI: median fluorescent intensity. e,f, Metabolic measurements from TFEB-GFP- or GFP-expressing BMDMs, showing (e) extracellular acidification rates (ECAR) and (f) oxygen consumption rates (OCR). Bars show mean ± s.d. from n = 7 independent biological replicates. Statistics derived from two-sided, unpaired Student’s t-tests. n.s. for P > 0.05. g,h, TCA cycle fueling determined by metabolic labelling of TFEB-GFP- and GFP-expressing BMDMs with ¹³C-glucose, or ¹³C-glutamine, or ¹³C-palmitate in the presence or absence of LPS/IFNγ. Results are based on GC-MS/MS measurements. (g) Bar graphs present fractional label of ¹³C-glucose in citrate, ¹³C-glutamine in succinate, and ¹³C-palmitate in citrate. Bars show mean ± s.d. from (left and middle panel) n = 6 and (right panel) n = 5 (GFP) and n = 6 (TFEB) biologically independent samples. (h) Itaconate-fueling from ¹³C-labelled glucose, glutamine and palmitate in 6 h LPS/IFNγ-treated BMDMs relative to naïve BMDMs. Bars show mean ± s.d. from n = 3 biologically independent samples.

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