Fig. 2: G6PD is enriched in old peroxisomes and influences the fate of daughter cells after asymmetric cell division.
a Age-selective isolation of peroxisomes and their proteomic analysis using mass spectrometry. Volcano plot of proteins differentially enriched between old and young peroxisomes. Significantly enriched proteins are marked in red (enriched with old peroxisomes) and green (enriched with young peroxisomes). b Immunofluorescent labeling of G6PD in hMECs with age-selectively labeled peroxisomes shows colocalization of G6PD with peroxisomes. G6PD is significantly enriched on old peroxisomes. Data shows 38 individual cells (unfilled circles) from three independent experiments (filled circles), p-value from one-way ANOVA test of independent experiments. c Representative immunofluorescence images show localization of G6PD (green) over-expressed in cytosol (cG6PD), in the peroxisomal matrix (pmtxG6PD) using a PTS1 targeting signal, and on the peroxisomal membrane (pmembG6PD) using a YgiM tail anchor. PMP70 immunofluorescent labeling show peroxisomes. Overexpression of G6PD on the peroxisomal membrane, but not in the peroxisomal matrix or in the cytosol, increases mammosphere formation of hMECs significantly. Data from eight independent experiments, p-value from one-way ANOVA test. d Schematic showing the G6PD activity and peroxisomal ether lipid synthesis pathway. NADPH produced by G6PD activity on the peroxisomal membrane is potentially utilized for peroxisomal ether lipid synthesis, and inhibiting AGPS can be employed as a strategy to probe the function of pmembG6PD on peroxisomal ether lipid synthesis. e Mammosphere formation by cG6PD and pmembG6PD after continuous inhibition of AGPS. AGPS inhibition reduces mammosphere formation and mitigates the boost induced by pmembG6PD. Data from five experiments, p-value from paired, two tailed t-test and one-way ANOVA test. f Organoid formation and branching efficiency of mMECs after continuous inhibition AGPS activity. Inhibiting peroxisomal ether lipid synthesis reduces organoid formation and branching efficiency of mMECs. Data from four biological replicates, p-value from paired, two tailed t-test. Data are presented as mean ± SD. Source data are provided as a Source Data file.
