XPR1 expression levels inversely correlate with polyP content in cells. HEK293 cells and MEG-01 megakaryocytes were transiently transfected with the indicated amounts of pCHIX-XPR1 or empty (Mock) vector. HEK293 (A) and MEG-01 (B) cells were analyzed by western blot with anti-XPR1 antibodies (top) after 24 and 48 hours, respectively. The cytoskeleton protein vasodilator-stimulated phosphoprotein (VASP) served as the loading control (bottom). XPR1 and polyP levels in XPR1-overexpressing HEK293 (C) and MEG-01 (D) cells. HEK293 (E) and MEG-01 (F) cells were transfected with 250 nM XPR1 siRNA and XPR1 mRNA expression and polyP were analyzed every 24 hours for 5 days. The expression of XPR1 mRNA was normalized to 18S rRNA signal and plotted, as a percentage of XPR1 expression at day 0 after control siRNA treatment (100%). Data are expressed as the mean ± standard error of the mean, by 1-way analysis of variance and Tukey’s multiple comparison test. *P < .05; **P < .01. PolyP from XPR1-overexpressing (OE) (G) or siRNA-treated XPR1 knockdown (KD) (H) cells was extracted, and equal amounts (10 ng per lane) of polyP were loaded, separated on polyacrylamide/urea gel, and visualized by negative DAPI staining. For the control, purified polyP was loaded before (−) or after (+) incubation with PPX (10 μg/ml for 1 hour).