Figure 7
Rescue of Tal1 expression during hemogenic endothelium culture allows the formation of Pre-HPCs and HP from endothelium. (A) Western-blot (WB) analysis showing the expression of transgenic HA-TAL1 in day 3 Dox Unt and Dox Dox i3TFs Tal1Δ/Δ hemangioblast cultures. The WB was performed on two independent repeats for each cell line. B-ACTIN was used as a loading control. (B) Representative flow cytometry analysis of i3TFs Tal1Δ/Δ Unt Unt, Dox Unt and Dox Dox hemangioblast cultures at day 3. The square highlights the endothelial cell population (VE-CAD+CD41−) sorted from the Dox Unt condition for subsequent hemogenic endothelium culture and RNA-seq. (C) RNA-seq (n = 4) analysis showing the expression of the indicated genes in the FACS sorted endothelial cell population. (D) Results of hemogenic endothelium cultures. Top: panels showing one representative flow cytometry analysis. Bottom: box plots summarizing four independent experiments. Significance was determined by Analysis of Variance (ANOVA) test. Error bars correspond to standard deviations. (E) Box plots summarizing the expression of CD45 in untreated and dox-treated HE cultures subjected to a liquid hematopoietic cell growth assay in the absence of doxycycline for 10 days. Whole i3TFs Tal1Δ/Δ Dox Dox hemangioblast cultures were used as a positive control. Significance was determined by Analysis of Variance (ANOVA) test. Error bars correspond to standard deviations. Unt Unt = untreated; Dox Unt = doxycycline added at EB day 2; Dox Dox = doxycycline added at EB day 2 and at day 0 of hemangioblast culture; CD45 = pan-hematopoietic marker.
