Fig. 4: ADIPINT knockdown alters mitochondrial pyruvate carboxylase abundance and interactome.

a PC enzymatic velocity measured through a change in absorbance of NADH (340 nm) after transfection with GapmeR NC (black) or ADIPINT-targeting GapmeRs 1–3. Velocity was measured over 20 min (n = 6-8). b Western blot quantification of PC protein and α-tubulin (housekeeping protein) in the same sample PC activity was measured. PC expression was normalized to α-tubulin. One representative western blot for PC and α-tubulin is given above (n = 6–8). c PC activity (measured as the time to 50% completion of the activity assay) expressed per amount of PC protein. PC activity and protein were normalized within each experiment and each data point represents an individual experiment (n = 5–6). d Intracellular oxaloacetate (n = 4) and e pyruvate levels (n = 8) normalized per experiment after ADIPINT knockdown using GapmeRs 1-3. a–e Target and control GapmeRs were compared by one-way ANOVA with Dunnett’s post-hoc test to assess significance. f Representative western blot for PC, the mitochondria-localized proteins TOM20 and Glutaminase (GLS) and the cytoplasmic protein GAPDH after cellular fractionation into nuclei, mitochondrial/membrane and cytoplasmic fractions. g Quantification of PC abundance in input and mitochondria/membrane fractions normalized to TOM20 (n = 5) and h GLS (n = 5). i PC activity measured in the mitochondria/membrane fraction after ADIPINT knockdown using GapmeR 1 (n = 4). Unpaired two-tailed t-test was used to compare significance between GapmeR 1 and Gapmer NC samples in the input and mitochondrial fractions. a–e and g–i each data point represents an independent experiment. j PC immunoprecipitation in the mitochondria/membrane fraction after ADIPINT knockdown (GapmeR 1) and compared to control (GapmeR NC) cells. Pathways enriched by proteins significantly altered between the two are conditions are displayed. k Proteins detected that map to the wikipathways Glycolysis and Gluconeogenesis (Homo sapiens) pathway are displayed with the log abundance values for each protein and each replicate. Proteins significantly reduced in PC immunoprecipitation samples after ADIPINT knockdown are marked with a #. Data are presented as mean values + /− SD for n < 6 and SEM for n ≥ 6. *p < 0.05, **p < 0.01, ***p < 0.001, ^#p < 0.001, ****p < 0.0001. Source data are provided as a Source Data file.