Inhibition of RNA Pol I by CX-5461 decreases the proliferation of immature erythroblasts. Human primary erythroblasts were derived from CD34⁺ progenitors cultured for 6 days with SCF, IL-6, IL-3, and dexamethasone; for 1 day with SCF, IL-6, and IL-3; for 4 days between days 7 and 11 with SCF and EPO; and then with EPO alone. CX-5461 (50 nM) was added to erythroblast cultures at day 9. (A) Quantification by qRT-PCR of pre-rRNA 45S transcript after 48 hours of treatment with CX-5461. Transcripts amounts were normalized to B2M, UBC, and ACTB and expressed as the normalized relative quantity (NRQ). Mean NRQ ± SEM of 3 experiments. (B) Percentage of RP neosynthesis after SILAC labeling of human erythroblasts for 48 hours in the presence or absence of CX-5461. Results are expressed as the mean ± SEM of 3 experiments. (C) Comparison of the ratios of non-RP, RPS, and RPL neosynthesis after SILAC between CX-5461-treated and untreated cells. Results are expressed as means ± SEM of 3 independent experiments. (D) Polysome profiling. Ribosomes from human erythroblasts, treated or not with CX-5461 for 48 hours, were purified on sucrose gradient, and the relative abundance of free-RNP complexes; the 40S, 60S, and 80S subunits; and polysomes were measured by absorbance at OD 260 nm. (E) Label-free proteomic for absolute quantification of RPs by mass spectrometry after 48 hours of incubation, with or without CX-5461. Results are expressed as the mean copy number ± SEM of 5 independent experiments. (F) Proliferation rate of human erythroblasts expressed as the cumulative number of cells at each time point. Mean ± SEM of 4 experiments. (G) Percentages of GlyA⁺ cells were quantified by flow cytometry and are shown as the mean ± SEM of 4 experiments. (H) Proportions of erythroid precursor populations at day 13 by cytological examination of May-Grünwald-Giemsa–stained cytospins. Mean percentages ± SEM of 4 experiments. (I) Flow cytometry of α4 integrin/CD49d and Band3 expression in GlyA⁺ cells identifying proE (I), baso1 (II), baso2 (III), polyE (IV), and orthoE (V) cells according to Hu et al.³  Absolute number of each precursor is shown as the mean ± SEM of 3 experiments. (J) Timeline of sorting of GlyA⁻ (progenitor) and GlyA⁺ immature precursors (proE) at day 8. CX-5461 (50 nM) was added at day 9 for 48 hours, and the cells were analyzed at day 11. (K) Absolute number (means ± SEM of 3 experiments) of unsorted cells (left), progenitors (progs; middle), and proEs (right). (L) Flow cytometry histograms (side scatter [SSC]/forward scatter [FSC]) for percentages of viable cells (means ± SEM of 3 experiments). (M) Flow cytometry histograms of GlyA expression (left) expressed as mean percentages ± SEM of GlyA⁺ cells (right). (L-M) Control: red bars; CX-5461: blue bars. (N) Colony assays. Cord blood–derived CD34⁺ progenitors were seeded for 14 days in methylcellulose with increasing concentrations of CX-5461 (left). Colony area and counts were determined and are represented as means ± SEM of 3 independent experiments (right). For all panels: *P < .05; **P < .01; ***P < .001; ****P < .0001, by Student t test.