Fig. 2. Dohh KD decreased protein synthesis and mitochondrial function in hepatic cells.

AML12 cells were transfected with 20 nM of negative (siNeg), Dohh (siDohh), or eIF5A (siEif5a) siRNA for 48 h. Puromycin (1 µg/ml) was added 1 h before harvesting cell lysate. a Western blot and densitometric analysis of eIF5A^H, and proteins after puromycin incorporation. (n = 3). b Western blot and densitometric analysis of Tfam, PGC1α, and mitochondrial proteins. (n = 3). c Proteomics and gene ontology (GO) enrichment analysis (with corrected p value indicated on the bar) of downregulated proteins in Dohh KD (siDohh) vs control AML12 cells. A Bonferroni correction was applied to correct for multiple testing. d Agilent Seahorse XF Mito Stress Test measurement of mitochondrial OXPHOS. Oligomycin (oligo), FCCP, rotenone (R), and antimycin A (A) were used at 1 µM each. (n = 15). Line graph is presented as mean value ± SEM. e Mitochondrial DNA copy number in Dohh or eIF5A knockdown cells. (n = 4). a, b, d, e Data were shown as box-and-whisker with median (middle line), 25th–75th percentiles (box), and min-max values (whiskers). Significance was calculated by one-way ANOVA or Kruskal–Wallis test, as appropriate. Source data are provided as a Source Data file.