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. 2016 Apr 21;6(5):692–703. doi: 10.1016/j.stemcr.2016.03.006

Figure 2.

Figure 2

cAMP-Mediated HSC-like Upregulation Occurs through the cAMP-Epac Axis

(A) Quantification of HSC-like frequency in viable (at day 14) after PKA or Epac inhibition. Data represent mean ± SEM of three independent experiments. Statistical analysis was performed using the t test. ∗p < 0.05, ∗∗p < 0.01; n.s., not significant.

(B) Representative flow cytometry plots (biexponential axis) showing the CD43+ blood cells and HSC-like cells (day 14) generated after forskolin + IBMX-mediated cAMP induction and PKA or Epac inhibition (PKAi, Epaci), with or without forskolin + IBMX.

(C) Quantification of radial spread of EBs (distance between center of EB and outer edge of the cellular spread, day 13). Data represent mean ± SEM of 100 EBs from three independent experiments. Statistical analysis was performed using the t test. ∗∗∗∗p < 0.0001; n.s., not significant.

(D) Analysis of hemogenic endothelial (CD43−CD34+CXCR4−CD73−VEcad+) phenotype (day 10), after cAMP induction and PKA or Epac inhibition (PKAi, Epaci) with or without cAMP induction. Data represent mean ± SEM of three independent experiments; mean fold change respective to control is shown. Statistical analysis was performed using the t test. ∗p < 0.05, ∗∗p < 0.01; n.s., not significant.

(E) Analysis of general (non-hemogenic) endothelium (CD43−CD34+VEcad+) at day 10, after cAMP induction and PKA or Epac inhibition (PKAi, Epaci) with or without cAMP induction. Data represent mean ± SEM of three independent experiments; mean fold change respective to control is shown. Statistical analysis was performed using the t test. ∗p < 0.05, ∗∗p < 0.01; n.s., not significant.

(F) Analysis of arterial endothelium (CD43−CD34+CD90+CD73+CXCR4+) at day 10, after cAMP induction and PKA or Epac inhibition (PKAi, Epaci) with or without cAMP induction. Data represent mean ± SEM of three independent experiments; mean fold change respective to control is shown. Statistical analysis was performed using the t test. ∗∗p < 0.01; n.s., not significant. For (D)–(F) the analysis was done at day 10, as the hemogenic endothelium is less abundant after this time point.

See also Figure S2.