Extended Data Fig. 3: Expression of CCND3 in erythroid cultures.
From: Reduced cyclin D3 expression in erythroid cells protects against malaria
a,b, Normalized expression levels (fragments per kilobase of transcript per million mapped reads; FPKM) of CCND3 in proerythroblast (ProE1, n = 3; ProE2, n = 3), basophilic erythroblast (BasoE, n = 4), polychromatophilic erythroblast (PolyE, n = 4), orthochromatophilic erythroblast (OrthoE, n = 4), and orthochromatophilic / reticulocyte (OrthoE-Ret n = 4) RNA-seq data from Ludwig et al.20 (a); and in proerythroblast (ProE, n = 3), early basophilic erythroblast (Early BasoE, n = 3), late basophilic erythroblast (Late BasoE, n = 3), polychromatophilic erythroblast (PolyE, n = 3), and orthochromatophilic erythroblast (OrthoE, n = 3) RNA-seq data from An et al.19 (b), reprocessed according to the Methods. Box-and-whiskers plots indicate the median (by a line across the box); the box ranges from the first quartile to the third quartile of the distribution; the “whiskers” on box plots extend from the box to the most extreme data points. Red dotted boxes indicate basophilic erythroblasts, characterized by the peak of CCND3 expression, and polychromatophilic erythroblasts, marked by the decline in CCND3 expression. c, Representative flow-cytometry dot plots of GPA+ gated erythroblasts showing expression of Band3 and integrin α4 at two successive points day 7 (left) and day 9 (right) of differentiation. d, Morphological appearance of cells collected at the same time points as above (day 7 and day 9) showing the prevalence of BasoE and PolyE stages. Representative cytospin slides were stained with May–Grünwald–Giemsa (n = 3 biologically independent experiments). Bars represent 10 μm. e, Relative expression of CCND3 mRNA quantified by RT–qPCR in different samples (n = 3 biologically independent experiments; 3 donors) for two time points, day 7 and day 9, of erythroid differentiation relative to a β2-microglobulin (β2M) endogenous control. Data are shown as box-and-whisker plots, with boxes indicating the interquartile range (IQR; 25th–75th percentile), the horizontal line the median, and whiskers extending to 1.5× IQR, and individual points beyond the whiskers shown as outliers. The two-sided P-value (P = 2.88−04) assessing the differential expression at the time points considered was obtained using the Wilcoxon–Mann–Whitney non-parametric test. The statistical results for all comparisons are provided in Supplementary Table 3.
