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. 2014 Oct 7;9(10):e110101. doi: 10.1371/journal.pone.0110101

Figure 1. ADP down-regulates nucleolin protein level.

Figure 1

(A) Dose-response. HUVEC were treated with the indicated concentrations of ADP for 72 h. Medium was replaced with fresh medium containing ADP every day. The nucleolin protein was detected by western blot. β-actin protein was detected as a loading control. (B) Time course. HUVEC were treated with 100 µM ADP for the indicated time periods. Medium was replaced with fresh medium containing ADP every day. The nucleolin protein was detected by western blot. β-actin protein was detected as a loading control. (C) Intracellular location of nucleolin. HUVEC were transfected with GFP-nucleolin-expression plasmid. G418-resistant cells were selected and GFP-nucleolin was detected by fluorescence microscopy. Cells were counterstained with 50 µg/ml Propidium Iodide (PI) to show nucleus staining. (D) ADP down-regulated over-expressed nucleolin. Nucleolin-over-expressed HUVEC were treated with 100 µM ADP for 72 h. Medium was replaced with fresh medium containing ADP every day. The GFP-nucleolin was detected by fluorescence microscope. Cells, mock-transfected with GFP-expression plasmid, were treated as control. (E) The effect of ADP on nucleolin mRNA levels in HUVEC. Cells were treated with the indicated concentrations of ADP for 72 h. Medium was replaced with fresh medium containing ADP every day. The mRNA level of nucleolin was detected by qRT-PCR. * P<0.05 compared with the control group. (F) The effect of ADP on nucleolin mRNA level in HAEC. Cells were treated with 100 µM ADP for the indicated time periods. Medium was replaced with fresh medium containing ADP every day. The mRNA level of nucleolin was detected by qRT-PCR. * P<0.05 compared with the control group. (G) The effect of ADP on nucleolin protein level primary human aortic endothelial cells (HAEC). (H) The effect of ADP, UDP, UTP, and ATP on nucleolin expression. Nucleolin-over-expressed HUVEC were treated with 100 µM ADP, or UDP, or UTP, or ATP for 72 h. Medium was replaced with fresh medium containing nucleotide every day. The GFP-nucleolin was detected by fluorescence microscope.