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. 2012 May 24;120(2):386–394. doi: 10.1182/blood-2011-12-399667

Figure 4.

Figure 4

EP-induced cell death is iron dependent. (A) HL60 cells were untreated or treated with 5 μg/mL of TPO, 25 ng/mL of EP, or 100u DFO for 72 hours preloaded or not with 500 μg/mL of ferric ammonium citrate (FAC) for 24 hours. Cell viability was measured by cell counts with trypan blue exclusion (top panel) and MTS assays (bottom panel) performed at 72 hours. Data represent the means ± SD of viable cells performed in triplicate. P values represent the difference between iron-loaded and noniron-loaded cells. *P < .05; **P < .01. (B) URE−/− cells were untreated or treated with 5 or 10 μg/mL of EP for 72 hours preloaded or not with 500 μg/mL of ferric ammonium citrate for 24 hours. Cell viability was measured by cell counts with trypan blue exclusion (top panel) and MTS assays (bottom panel) performed at 72 hours. Data represent the means ± SD of viable cells performed in triplicate. P values represent the difference between iron-loaded and noniron-loaded cells. *P < .05; **P < .01; ***P < .001. (C) HL60 Cells were incubated in 10μM Cell Tracker Orange for 30 minutes, washed, and analyzed by FACS. Cells were treated without EP, with 5 μg/mL of EP, or were preloaded with 500 μg/mL of ferric ammonium citrate and then treated with or without 5 μg/mL of EP. FACS analysis was performed at 72 hours. The percent change in FACS MFI ± SD (n = 3) of Cell Tracker Orange labeled HL60 cells treated with 5 μg/mL of EP with or without iron preload relative to untreated cells, corrected for hours 0 MFI. The P value represents the difference between the MFI of the iron-preloaded cells treated with EP and the cells treated with EP without iron preload. *P < .05; **P < .01. EP slows cell division in HL60 cells and cell division is rescued by preloading cells with iron. (D) Representative morphology of HL60 cells treated for 72 hours under the above conditions shown at 20× (left panel) and 63× (right panel). HL60 cells preloaded with iron and subsequently treated with EP display less segmented nuclei than EP-treated cells without iron (cells with increased nuclear segmentation are indicated by arrows). (E) FACS analysis of CD11b expression (i) in HL60 cells treated with 5 μg/mL of EP (orange line) versus cells preloaded with 500 μg/mL of ferric ammonium citrate (FAC) for 24 hours and then treated with 5 μg/mL of EP (blue line) for 72 hours. FACS analysis of CD14 expression (ii) in HL60 cells treated with 5 μg/mL of EP (orange line) versus cells preloaded with 500 μg/mL of FAC for 24 hours and then treated with 5 μg/mL of EP (blue line) for 72 hours. CD11b and CD14 are overexpressed with EP treatment and rescued by preloading cells with iron. The fold change of FACS MFI ± SD (n = 3) of CD11b expression (iii) and CD14 (iv) in HL60 cells preloaded with 500 μg/mL of FAC and then treated with 5 μg/mL of EP compared with cells treated with 5 μg/mL of EP without iron load is shown. P values represent the difference between iron-loaded and noniron-loaded cells. *P < .05; **P < .01.