Figure 2.

Hdac1 or Hdac2 Deletion Reduces Hematopoiesis

(A) Western blot on HE (CDH5⁺/CD41⁺) cells derived from control and Hdac1- or Hdac2-deleted cultures. β-Actin was used as a loading control.

(B) Scheme of the experimental setup. ESCs were differentiated to EBs before isolation of FLK1⁺ cells. Induction of Hdac1 or Hdac2 deletion was induced at this stage by addition of 4-hydroxy-tamoxifen (1 μM) in Li-Blast culture. Cultures were analyzed at days 1 and 3 by FACS. At day 3, a fraction of the cultures were also re-plated into CFU-C assays.

(C) Representative FACS analysis at days 1 and 3 of Hdac1lox/lox and Hdac1Δ/Δ cultures. Cells were stained for the endothelial marker CDH5 and the hematopoietic marker CD41. The blue boxes indicate the CD41⁺ (CDH5^–) gate used in the bar chart.

(D) Bar chart quantifying the percentage of CD41 (CDH5^–)- and CD45 (CDH5^–)-positive cells generated at days 1 and 3 of Li-Blast from Hdac1lox/lox and Hdac1Δ/Δ cultures. Mean of three independent experiments (n = 3) are shown and p values were calculated with a paired t test.

(E) CFU-C colony assay of day 3 Hdac1lox/lox and Hdac1Δ/Δ cultures. Mean of three independent experiments (n = 3) are shown with p values calculated with a paired t test.

(F) Representative FACS analysis at days 1 and 3 of Li-Blast of Hdac2lox/lox and Hdac2Δ/Δ cultures. Staining for the endothelial marker CDH5 and for the hematopoietic marker CD41 is shown. The blue boxes indicate the CD41⁺ gate used in the bar chart.

(G) Mean percentage from three independent experiments (n = 3) of the frequencies of CD41 (CDH5^–)- and CD45 (CDH5^–)-expressing cells at days 1 and 3 of Hdac2lox/lox and Hdac2Δ/Δ cultures are shown. The p values were calculated with a paired t test.

(H) Day 3 Hdac2lox/lox and Hdac2Δ/Δ Li-Blast cultures were tested for CFU-C activity. Mean colony numbers from three independent experiments (n = 3).

Error bars depict SEM. The p values were calculated with a paired t test.