Figure 1.

Pharmacologic Treatment with a Combination of EZH2 and HDAC Inhibitors Enhances ECFC Migration, Capillary Network Formation, and Resistance to Serum Starvation-Induced Apoptosis

(A) GSK-343 and panobinostat lead to a global decrease in H3K27me2/3 levels and an increase in H3/H4-acetyl levels, respectively, as analyzed by western blot. Molecular masses are indicated in kDa. Total histone H3 and H4 serve as loading controls.

(B) GSK-343 and panobinostat do not affect the overall level of indicated proteins, as analyzed by western blot. Molecular masses are indicated in kDa. Tubulin serves as a loading control.

(C) GSK-343 and panobinostat increase the kinetics of ECFC migration as measured by gap closure assay. Left: representative pictures of ECFCs stained with crystal violet after 10 hr migration (10× magnification; scale bar, 250 μm). Dashed circles represent the gap area prior to cell migration. Right: fraction of the gap area invaded by migrating cells at the indicated times. Average from thee independent experiments (n = 3) each performed in duplicate are shown ±SEM.

(D) GSK-343 and panobinostat increase ECFC-mediated capillary-like structure formation on Matrigel. Left: representative pictures of capillary-like structures stained with calcein (2.5× magnification; scale bar, 1 mm). Right: total capillary length of the network is expressed as the mean percentage of control values corresponding to cells treated with vehicle ±SEM. Data shown from three independent experiments (n = 3) each performed in duplicate.

(E) GSK-343 and panobinostat do not induce apoptosis in ECFCs as measured by fluorescence-activated cell sorting (FACS) after 7-AAD and annexin V staining.

(F) GSK-343 and panobinostat reduce serum starvation-induced apoptosis in ECFCs as measured by FACS after 7-AAD and annexin V staining. ECFCs were grown in serum-containing EGM-2 medium or serum-depleted EBM-2 medium.

(E and F) Data expressed as mean percentage of control values corresponding to cells treated with vehicle ±SEM from three independent experiments (n = 3), each performed in duplicate.

(G) GSK-343 and panobinostat decrease ECFC proliferation as analyzed by bromodeoxyuridine (BrdU) incorporation. The percentage of cells incorporating BrdU is indicated ±SEM from four independent experiments (n = 4), each performed in duplicate.

^∗∗∗p < 0.001; ^∗∗p < 0.01; ^∗p < 0.05; ns, non-significant. See also Figures S1 and S2.