Extended Data Fig. 5: Overexpression of SIRT6 WT negatively affects glycolytic gene expression, and slows down glycolytic flux, while minimally affecting the mitochondrial TCA cycle in HSC2 cells.

Extended Data Fig. 5 (related to Fig. 3). a, Chromatin fraction and whole cell lysates were extracted respectively and were analyzed by Western blot in doxycycline-inducible SIRT6 WT or H133Y overexpressing HSC2 cells in addition to EV (empty vector) control. b, Glycolysis stress test was performed using the Seahorse bioanalyzer following the manufacturer’s instruction in HSC2 cells pretreated with doxycycline for 24hrs. ECAR value was normalized by cell number of each well using the Cyquant cell proliferation assay kit. Data indicate mean ± S.D. Each dot represents one biologically independent sample and s.d. (n = 9 for S6HY and n = 16 for S6WT). Student’s t-tests were performed (two-sided). c,e, Relative enrichment of fully labeled glycolysis intermediates (c) or labeled TCA cycle intermediates (e) after incubation with U-¹³C-glucose at a given time point either in SIRT6 WT or H133Y overexpressing HSC2 cells (26hr post doxycycline). Data indicate mean ± S.D. Data are from three biological replicates. d, Annexin V and PI staining in HSC2 cells 24hr post dox, analyzed by MACSQuant VYB Data indicate mean ± S.E.M. Data are from three biological replicates. *** p<0.001.

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