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. 2014 Dec 9;125(6):930–940. doi: 10.1182/blood-2014-06-585513

Figure 5.

Figure 5

RUNX1 knockdown in H9 ESC line and in an FPD/AML iPSC line with monoallelic RUNX1 deletion leads to the same phenotype as the FPD/AML-DN iPSC line. (A-G) RUNX1 knockdown in H9 ESC line. (A) Analysis of RUNX1 protein expression in mesodermal population from control and shRUNX1_1 ESC lines. (B-C) CD34+CD43+ cells were sorted at day 11 and tested for their colony-forming potential in methylcellulose assay and fibrin clot culture. (B) Assessment of erythroid progenitors in methylcellulose culture. (C) Assessment of MK progenitors in fibrin clot cultures. (B-C) The histograms show 1 representative experiment of 4 for erythroid progenitors and of 3 for MK progenitors, each in triplicate. Error bars represent ± SD of triplicate. (D-E) Analysis of hematopoietic populations generated at day 18 from progenitors sorted at day 11. (D) Absolute number of generated erythroid cells (GPA+) normalized to 1 × 102 plated progenitors (n = 4). (E) Absolute number of generated MK cells (CD41+CD42+) normalized to 1 × 102 plated progenitors (n = 4). (D-E) The histograms show the average of 4 independent experiments. Error bars represent ± SD of average. (F) CD34+CD43+ cells were sorted at day 14 and were tested for their CFU-GM colony-forming potential in methylcellulose assay. The histograms show 1 representative experiment of 4, each in triplicate. Error bars represent ± SD of triplicate. (G) Analysis of hematopoietic populations generated at day 21 from progenitors sorted at day 14. Absolute numbers of generated monocytes and/or macrophages (CD14highCD15low; n = 4) and granulocytes (CD15high; n = 4) normalized to 1 × 102 plated progenitors. The histograms show the average of 4 experiments. Error bars represent ± SD of average. (H) RUNX1 knockdown in D(a) iPSC line with shRUNX1_1. CD34+CD43+ cells were sorted at day 14 and tested for their CFU-GM colony-forming potential in methylcellulose assay. The histograms show 1 representative experiment of 3, each in triplicate. Error bars represent ± SD of triplicate. (I-J) Transcriptome analysis of CD34+CD43+ progenitors derived from shControl and shRUNX1_1 transduced hESC line sorted at day 14 of culture. FC: fold change between cells transduced with shRUNX1_1 and shControl lentiviruses. Only genes with expression variation ± 1.5 and P < .001 (analysis of variance [ANOVA] test) are listed. (J) Variation in expression of genes involved in p53-dependent DNA damage response; P < .001 (ANOVA test). E, erythroid; G/M, granulo-monocyte; I, immunity; IN, inflammation; MK, megakaryocyte. *P < .05; **P < .01; ***P < .001, Student t test.