Figure 1.

RPS14 downregulation results in limited ribosome availability and reduced GATA1 translation. (A-C) Knockdown of RPS14 by shRNA in human primary erythroblasts from healthy donors. (A) Schematic workflow. (B) RSP14 and GATA1 protein expression determined by western blot. Actin was used as a loading control (n=3). (C) (Left) Representative immunohistochemical images of human bone marrow biopsies stained for GATA1 protein (brown) and counterstained with Hematoxylin & Eosin in del5q patients (n=3) and normal healthy controls (n=3). (Right) A comparison of single cell saturation intensity between patients and healthy controls. A Mann-Whitney test was used to calculate P-value. (D-L) Knockdown of RPS14 in the UT-7/EPO cell line via inducible shRNA (sh640 and sh641). (D) Schematic workflow. (E) GATA1 protein expression determined by western blot. Signals were quantified by normalization to actin (n=3). (F) Transcriptome analysis of UT-7/EPO shRPS14 cell lines. Gene set enrichment analysis (GSEA) shows variations in GATA1 targets. (G) Bioanalyzer quantification of 18S and 28S rRNA expressed as mean ratios±standard error of mean (SEM) (n=3). (H) Quantitative proteomic analysis of ribosomal protein (RP) from whole cell lysates. Data are expressed as a fold change in the copy number per cell between shRPS14 and shSCR conditions in doxycycline-induced cells (n=3). (I) Absolute quantification of translation by quantification of O-propargyl-puromycin (OPP) incorporation per cell by flow cytometry, shown by the mean fluorescence intensity±SEM (n=3). (J) Polysome profiling. Free 40S, free 60S and ribosome 80S (fraction I), light and medium polysomes (fraction II), and heavy polysomes (>5 ribosomes; fraction III) are indicated. (K and L) Relative distribution of GATA1 and MYC mRNA in ribosomal fractions. Quantification values determined by real-time-quantitative polumerase chain reaction are expressed as mean±SEM (n=3). Unpaired two-tailed Student t-test: *P<0.05; **P<0.01; ***P<0.001.