Fig. 5. CHAC1 supports the matrix cys under Cys₂ deprivation.

a Schematic workflow of mitochondrial isolation coupled to LC-MS for the compartmentalized detection of thiol-containing metabolites; Mito IP, mitochondrial immunoprecipitation. NEM, n-ethylmaleimide. b Determination of the half-life of cytosolic and matrix Cys in H1299-HA-Mito and H2009-HA-Mito cells cultured in the absence of extracellular Cys for 20 h (n = 3 biologically independent samples per time point). c Determination of the half-life of cytosolic and matrix GSH in H1299-HA-Mito and H2009-HA-Mito cells cultured in the absence of extracellular Cys for 20 h (n = 3 biologically independent samples per time point). d Immunoblot analysis of NFS1, CHAC1, and β-Actin expression in cystine-fed or starved H2009-HA-Mito cells 3-days post-lentiviral infection with either a scramble control or NFS1-targeting hairpin that were also subject to 5-days of 0.2 μg/mL doxycycline treatment to induce shRNA-mediated knockdown of CHAC1. e Matrix cysteine levels in H2009-HA-Mito cells deficient in CHAC1 and or NFS1 that were subjected to 2 h of cystine starvation (n = 4 biologically independent samples per condition, except when n = 3 for shREN + scramble and shCHAC1 #3 + shNFS1). f Matrix GSH levels in H2009-HA-Mito cells deficient in CHAC1 and or NFS1 that were subjected to 12 h of cystine starvation (n = 4 biologically independent samples per condition). For (e, f) data are representative of the matrix pool of each metabolite relative to cystine replete conditions. Data represent mean values ± s.d. For (e, f) P values were calculated using a two-way ANOVA with a Šidák’s multiple comparisons test. a created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license, https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en. For (b–f), source data provided as a Source Data File.