Fig 1: Suppression of OXPHOS leads to ribosomal defects(A) Schematic illustration of transcriptome analysis of differentiated HSPCs with OXPHOS inhibition. Differentiated cells toward erythroid lineage on days 3 and 7 treated with or without Rot were collected and evaluated using bulk transcriptome sequencing. Ribosome biogenesis and globin biosynthesis are the representative biological events that occurred on days 3 and 7 of erythropoiesis, respectively.(B) Enrichment analysis of GO terms enriched by up- and downregulated DEGs in differentiated HSPCs on days 3 and 7 and DBA patient samples, respectively. The biological pathways or events, including OXPHOS and ribosome biogenesis, were suppressed and enriched in differentiated HSPCs with OXPHOS inhibition and DBA samples.(C) Representative Agilent Bioanalyzer 5400 electrophoretogram profiles of total RNA from differentiated erythroid cells on Day 7. rRNA species were disturbed in differentiated HSPCs treated with different concentrations of Rot. The highlighted areas indicate altered rRNA species (reduced 18s rRNA: red; newly synthesized rRNA species: dark blue and red arrows) in ribosomes in differentiated erythroid cells with OXPHOS inhibition.(D) Comparison of polysome profiling between differentiated erythroid cells with or without 0.1 μM Rot treatment.(E) Real-time PCR analysis indicating a significant overall reduction in the expression of most DBA-causative genes in differentiated HSPCs with OXPHOS inhibition on Day 7. ACTIN was used as the reference gene. Data are represented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01.(F) Integrative analysis of proteome and transcriptome data revealing the overall reduction of all constitutive ribosomal proteins at both the protein and mRNA levels. OXPHOS suppression leads to the comprehensive reduction of ribosomal proteins in differentiated erythroid cells.(G) Real-time PCR analysis indicating a decrease in the gene expression of RPS6 in differentiated HSPCs with OXPHOS inhibition on Day 7. GAPDH was used as a reference gene in real-time PCR assays. Data are represented as mean ± SEM. ∗p < 0.05; ∗∗∗p < 0.001.(H) Immunofluorescence analysis of RPS6 revealing defects in ribosome levels in differentiated HSPCs with OXPHOS inhibition on Day 7. Scale bar: 60 μm. See also Figure S2.