Figure 6

Activation of the CD34⁺PRLR⁺ progenitors by prolactin. UCB-derived CD34⁺ HSCs were expanded for 9 days using Flt3L, LDL, SCF and TPO, and CD34⁺PRLR⁺ cells were sorted using FACS. (A) Quantitative PCR and expression of PRL mRNA in CD34⁺PRLR⁺ cells is shown relative to its expression in CD34⁺PRLR⁻ cells after normalizing to the expression of GAPDH (n = 4/group). (B) RNAseq, principal component analysis of global gene expression and PCA plot is shown (n = 3). Prolactin treated (Red) and non-treated (Blue) were shown for donors 42, 74 and 75. (C) Heat map showing relative expression of PRL-regulated downstream genes found in the IPA causal network list (n = 3/group). (D) IPA generated network highlighting the relationship between PRL, SMAD7, and TGFβ-1. Values represent the log2 fold change between PRL treated and untreated samples (n = 3/group). (E) Sorted 9 days old CD34⁺PRLR⁺ cells were treated with prolactin or a control, PBS, for 48 hours and expression level of SMAD7 in control or treated cells was analyzed using western blot. Bar graph shows quantification of SMAD7 levels and mean expression in each condition. One representative western blot showing SMAD7 and the loading control, α-tubulin (n = 5). Both SMAD7 and α-tubulin were probed from the same gel/membrane. (F) ELISA and the concentration of TGFβ-1 in the supernatant of prolactin treated or control CD34⁺ culture is shown in bar graph (n = 4/group). (A,E,F) Data is shown as means ± SD, paired t-tests and significance is depicted (* = p < 0.05, ** = p < 0.01).