Fig. 4: Untreated DBA erythroid progenitor cells are forced to progress into the cell cycle.

a GSEA of G1/S phase transition in C4 between the NC and UT, and the UT and GCR groups. b Beeswarm plot showing the expression of the G1/S phase transition gene set in C3 among the samples. c CDK1 expression in C3 is shown among groups. d GSEA of regulation of DNA replication between the NC and UT and the UT and GCR groups. e Beeswarm plot showing the expression of the DNA replication gene set in C3 among the samples. f The expression of CDT1 in C3 is shown among groups. g The fraction of cells in G1, S and G2/M in C3 among groups. h Heatmap representing normalized enrichment score (NES) from GSEA for comparison between the indicated groups in C3 cells at the S-phase of cell cycle; the colored rectangles indicate significant enrichment (namely, higher expression) of corresponding processes in the designated groups (P < 0.05). i Immunofluorescence measuring DNA damage with γH2AX expression in RPS19-diminished primary erythroid cells on day 4 of differentiation with or without Dex treatment. j Quantification of immunofluorescent intensity of γH2AX. The signal intensity of RPS19- depleted cells was normalized to the scramble (Scr) control. k, l Western blot analysis showing the protein expression of RPS19 (k) and P53 (l) after lentiviral- mediated shRNA knockdown in erythroid cells derived from CB-CD34⁺ cells on day 8 of differentiation. α-tubulin serves as the loading control. n = 3 independent experiments. m Quantification of immunofluorescent intensity of γH2AX in erythroid cells derived from CB-CD34⁺ cells on day 8 of differentiation. The signal intensity of each group (RPS19 single, P53 single or their double depleted cells) was normalized to the corresponding control. For (j and m), results are presented as the mean ± SEM. P values were determined using Student’s t test, ***P < 0.001; *P < 0.05;ns, no statistical significance. n ≥ 3 independent experiments. For all the beeswarm plots (b, c, e and f), each dot represents the expression value for each single cell that was calculated by summing the log₂ transformed UMI of every gene within the gene set. Diamonds represent mean expression values for each cluster, and boxes represent the median and quartiles. P values were determined using Wilcoxon rank sum tests. ****P ≤ 0.0001, ***P ≤ 0.001 and **P ≤ 0.01; ns, no significance. See also Supplementary Figs. S5,6 and Table S6.