Fig 2: DGUOK regulates NAD+ biosynthesis through NMNAT2. (A) Western blot analysis of NMNAT2 and NDUFB8 expression level in NMNAT2-KD and control H1650 cells. (B) LC-MS analysis of NAD+ level in NMNAT2-KD and control H1650 cells. (C) Quantification of NAD+ level in NMNAT2-KD and control H1650 cells. (D) Western blot analysis of NMNAT2, NDUFB8 and DGUOK expression levels in DGUOK-KO, DGUOK-KO/NMNAT2-OE and control H1650 cells. (E) LC-MS analysis of NAD+ level in DGUOK-KO, DGUOK-KO/NMNAT2-OE and control H1650 cells. (F) Quantification of NAD+ level in DGUOK-KO, DGUOK-KO/NMNAT2-OE and control H1650 cells. ***p < 0001.

Fig 3: DGUOK depletion inhibits lung adenocarcinoma CSC through an AMPK-YAP1 signaling axis AThe effects of DGUOK depletion on the YAP1 total protein levels and p-S127 YAP1 levels in H1650 cells, as determined by Western blotting.B, CThe effects of DGUOK depletion in H1650 cells on YAP1 nuclear translocation (B) and the mRNA transcript levels of YAP/TAZ target genes CTGF and CYR61 (C), as determined by immunofluorescence staining and qPCR, respectively.DThe effects of DGUOK depletion and compound C treatment on the levels of activated AMPK (pT172-AMPK), phosphorylated ACC (pS79-ACC), total YAP1 and pS127-YAP1 in H1650 cells, as determined by Western blotting.EThe effects of DGUOK depletion and compound C treatment on the phosphorylation status of YAP1, as determined by Phos-tag gel electrophoresis and YAP1 Western blotting.F, GThe effects of DGUOK depletion and compound C treatment on YAP1 nuclear translocation (F) and the mRNA expression of CTGF, CYR61 (G) in H1650 cells.HRepresentative IHC staining showing the expression of DGUOK and YAP1 in lung adenocarcinoma patient specimens.ICorrelation between DGUOK staining intensity and NDUFB8 staining intensity in a cohort of 113 lung adenocarcinoma patients. rs = 0.369, P < 0.0001, as determined by Spearman's correlation test.JExpression levels of YAP1 protein in control and DGUOK KO cells with or without ectopically expressed 6SA-YAP1 mutant, as determined by Western blotting.KThe effects of ectopic 6SA-YAP1 on lung tumor sphere formation abilities in control and DGUOK KO H1650 cells.LThe effects of ectopic 6SA-YAP1 in control and DGUOK KO H1650 cells on ALDH+ populations as determined by flow cytometric using ALDEFLUOR assay.Data information: Data are shown as mean ± SD. n = 3 independent replicates in (C, G and K). P values were determined by two-tailed, two-sample Student's t-test. Experiments in (D, E, and J) were repeated with two biological replicates with similar results; experiments in (A–C, F, G, L) were repeated with three biological replicates with similar results.Source data are available online for this figure.

Fig 4: Preserved mitochondrial OXPHOS complex activities in ADQ-/- hearts.Enzymatic activity of complex I subunit NDUFB8 (A), complex II subunit 30kDa (B), and complex IV subunit II (C) in isolated mitochondria of ADQ-/- and WT hearts; n = 5–6. * p<0.05 vs. WT.

Fig 5: Fascin and mtF-Actin Regulate the Biogenesis of Respiratory Complexes in NSCLC(A) Waterfall plot showing fold changes of 12 mitochondrial proteins differentially regulated in fascin KO H1650 cells. Fold changes (log2) are presented as mean ± SEM of results from two independent samples.(B and C) Western blotting showing the decrease in respiration Complex I subunits in fascin KO H1650 (B) and H23 (C) cells.(D and E) Western blotting showing the increase in the levels of Complex I subunits in H23 cells overexpressing wild-type fascin (D) but not the S39E and 149-151A mutants (E).(F) shRNA knockdown of INF2-CAAX abrogated fascin-mediated increase in mt-ND1 and NDUFB8 in H1650 cells.(G and H) The effect of fascin (G) and INF2-CAAX (H) depletion on the levels of respiratory super complexes in H1650 cells, as determined by BN-PAGE and western blotting using anti-OXPHOS antibody cocktails (recognized all 5 complexes) and antibodies specific for each respiratory complex (Cx I–V). S. C. indicates respirasomes consisting of Complex I, III, and IV.Representative results from at least three independent experiments are shown.