Measurement of PLA2 activity.

<p>(<b>A</b>) HL-60 cells were treated with 50 nM CHEC-9 or TBS vehicle for 4days with stimulation of PMA. The large increase of sPLA2 activity after differentiation was significantly attenuated by CHEC-9 treatment. (<b>B</b>) Differentiated SY5Y neuronal cells were subjected to medium change and serum deprivation for 2days. Compared with vehicle group, CHEC-9 treatments at 1 and 50 nM both reduced sPLA2 activity, but this change just missed significance (p = 0.07). (<b>C, D</b>) cPLA2 and iPLA2 activity of HL-60 cell homogenates. There was no difference between the undiffentiated and differntaited HL-60 cells. CHEC-9 treatment did not show inhibition of the cPLA2 or iPLA2 activities.</p

Protective effects of CHEC-9 on SY5Y cells.

<p>Retinoic acid differentiated SY5Y neuronal cells were subjected to different conditioned media for 72 hrs. <b>Cell process survival (A–F).</b> Long and elaborated processes were seen in cultures that were subjected to control media either from SY5Y cultures or undifferentiated HL-60 cultures (<b>A, B</b>). Dramatic degeneration of neuritic processes was found when SY5Y cells were subjected to medium from differentiated HL-60 cultures (<b>C</b>). These degenerative changes were reversed by CHEC-9 (50 nM, <b>D</b>). Medium from HL-60 cultures -pretreated with 50 nM CHEC-9 during differentiation, also preserved processes (<b>E</b>). (<b>F</b>) Quantification of the results. <b>Cell viability</b> (<b>G</b>). Both SY5Y conditioned medium and undifferentiated HL-60 did not affect SY5Y survival, whereas the medium from differentiated HL-60 cells significantly decreased viability. 50 nM CHEC-9 significantly restored the cell number to control level. Medium from HL-60 cultures-pretreated with 50 nM CHEC-9 during differentiation, also promoted cell survival. (* <i>p</i><0.05, ** <i>p</i><0.005).</p

Phagocytosis activity and cytokine levels of macrophages differentiated from HL-60 cells.

<p>Most macrophages in vehicle group were labeled with red beads and the intensity of beads was high (<b>A</b>). Macrophages treated with CHEC-9 engulfed significantly fewer beads after 4 days differentiation and resulted in a lower percentage of cells containing beads. Quantification of the results (<b>C, D</b>). Decreased expression of TNF-α and IL-6 by 50–60% was found in the cell medium of CHEC-9 treated group (<b>E</b>). (** <i>p</i><0.005).</p

Inhibition of differentiation of HL-60 cells by CHEC-9.

<p>HL-60 cells were treated with 50 nM CHEC-9 or TBS vehicle for 4days with stimulation by PMA. HL-60 cells adhere to plate surface, flatten out and grow processes (<b>A</b>). CHEC-9 treatment decreased the number of cells with process (<b>B</b>). Quantification of Coommassie blue staining (<b>C</b>). CHEC-9 did not affect viability of the cells (<b>D</b>). Immunostaining for CD36 showed that the fluorescent intensity of CHEC-9 group was decreased compared with vehicle group (<b>E, F</b>). This observation was supported by western analysis (<b>G</b>). Quantification was by normalization to GAPDH bands. (* <i>p</i><0.05, ** <i>p</i><0.005).</p

Cell cycle analysis using flow cytometry.

<p>(A) The representative FACS plots displayed differences in cell cycle phases of EPCs transfected with Ad-GFP, Ad-TM-FOXO3a or Ad-shRNA-FOXO3a. (B) Cell cycle analysis demonstrated that the proliferation index (the percentage of cells in S- and G2- phases) was significantly elevated after FOXO3a silencing when compared to the proliferation index in Ad-GFP group. Data were presented as the mean±SD of three independent experiments. *p<0.05 vs. Ad-GFP.</p

Representative microscopy of the morphology of EPCs at 24-GFP, Ad-TM-FOXO3a or Ad-shRNA-FOXO3a.

<p>Representative microscopy of the morphology of EPCs at 24-GFP, Ad-TM-FOXO3a or Ad-shRNA-FOXO3a.</p

EPCs were isolated and double-labeled for Dil-ac-LDL and UEA1.

<p>(A) EPCs(CD133⁺CD34⁺CD31⁺ cells) were isolated using fluorescence-activated cell sorting. (B)Fluorescence microscopy illustrates that endothelial progenitor cells are positive for Dil-ac-LDL(Red) and UEA1(Green) by HMC confocal microscopy.</p

Growth curve of EPCs within 72 hours after transfection.

<p>1.5x10⁵ EPCs were seeded in a 6-well plate and transfected with different recombinant adenovirus vectors. Cell number was counted by using a haemocytometer for a consecutive three days. Growth curve of EPCs showed cell growth pattern within 72 hours after transfection.</p

Protein expression of FOXO3a in EPCs transfected with Ad-GFP, Ad-TM-FOXO3a or Ad-shRNA-FOXO3a; meanwhile, untransfected EPCs were operated as a negative control.

<p>Blots were scanned, and expression of FoxO3a was quantified by densitometric analysis. The ratios for FoxO3a/Tubulin are shown. Data are mean±SE, n = 3, ^*p<0.05 vs. Ad-GFP or untransfected group.</p

FOXO3a modulated the expression of cell cycle regulatory proteins in EPCs.

<p>(A) EPCs were transfected with the indicated vectors. Cells were lysed and protein expression was determined by Western blotting. (B)Blots were scanned, and expression of protein was quantified by densitometric analysis. The ratios of CDK2, cyclinD1, P27 or PCNA to Tubulin are shown. Band intensity analysis was performed with Quantity One Software. Knockdown FOXO3a led to dramatic protein expression increases in CDK2, cyclinD1 and PCNA. Data are mean±SE, n = 3, *p<0.05 vs. Ad-GFP.</p