Extended Data Fig. 2: Mitochondrial NAD+ is limiting for PDAC cell proliferation under hypoxia.
(a) Immunoblot analysis of FLAG-tagged cytoplasmic (cyto-) and mitochondrial (mito-) LbNOX heterologous expression in the indicated cell lines. ACTB was used as a loading control. (b) Fraction of labelled aspartate (left) and citrate (right) derived from labelled glutamine in control and cyto- or mito-LbNOX expressing HY15549 cells cultured for 8 h with [U-13C]-Glutamine (500 μM) with piericidin treatment (50 nM) or under 0.5% oxygen. Colours indicate mass isotopomers. (c) Expression of LbNOX enzymes rescues proliferation of MIA PaCa-2 cells under piericidin treatment (50 nM), whereas only mito-LbNOX expression rescues proliferation under 0.5% O2. Data is shown as fold change in cell number in the indicated cell lines grown for 5 days relative to untreated cells cultured at 21% O2. (d) Fraction of 13C-labeled aspartate (m + 4) and malate (m + 4) in HY15549 parental and GOT2-knockout cells transduced with a control vector or an sgRNA-resistant GOT2 cDNA cultured for 24 hours under hypoxia in the presence of [U-13C]-L-glutamine (1 mM). (e) Relative fold change in cell number of HY15549 GOT2-knockout cells transduced with a control vector or the indicated cDNAs grown for 5 days under hypoxia (0.5% O2) to those cultured under normoxia (21% O2). b-e, Bars represent mean ± s.d. b-e, n = 3 biologically independent samples. Statistical significance was determined by two-tailed unpaired t-test.
