Figure 1.
ADP down-regulates nucleolin protein level.
(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.
Figure 2.
P2Y1, 12, and 13 receptors are not involved in ADP-induced nucleolin down-regulation.
(A, B) The mRNA levels of P2Y1, 12, and 13 receptors in HUVEC (A) and HAEC (B). Cells were treated with the indicated concentrations of ADP for 24 h. The mRNA levels of P2Y1, 12 and 13 receptors were detected by RT-PCR. β-actin transcript was detected as a loading control. (C) The mRNA levels of P2Y1, P2Y12, and P2Y13 receptors in 5–8F cells. (D) The effect of P2Y1 receptor inhibitor MRS2179 (M2179) on ADP-induced down-regulation of nucleolin protein. HUVEC, pre-treated with the indicated concentrations of MRS2179 for 1 h, were re-treated with 100 µM ADP for 72 h. The nucleolin protein levels were detected by western blot. β-actin transcript was detected as a loading control. (E) The effect of P2Y12 receptor inhibitor PSB0739 (PSB) on ADP-induced down-regulation of nucleolin protein. (F) The effect of P2Y13 receptor antagonist MRS2211 (MRS) on ADP-induced down-regulation of nucleolin. (G, H) The effect of MRS2211 on the nucleolin protein levels in HUVEC (G) and HAEC (H). (I, J) The effect of P2Y13 receptor agonist 2-MeSADP on nucleolin protein levels in HUVEC (I) and HAEC (J). (K) 2-MeSADP did not regulate over-expressed nucleolin protein. Nucleolin-over-expressed HUVEC were treated with the indicated concentrations of ADP for 72 h. The GFP-nucleolin expression was detected by fluorescence microscope.
Figure 3.
Nucleolin protein down-regulation is involved in ADP-induced cell cycle arrest in S phase.
(A) ADP induced cell cycle arrest in S phase. HUVEC were treated with the indicated concentrations of ADP for 72 h. Medium was replaced with fresh medium containing ADP every day. The detached cells were removed, and the adhered cells were collected for cell cycle test by PI staining. (B) Quantitation of cell cycle arrest in S pahse induced by ADP in (A). * P<0.05 compared with the control group. (C) ADP induced time-dependent cell arrest in S phase in HUVEC. * P<0.05 compared with the control group. (D) Nucleolin over-expression partly reversed ADP-induced cell cycle arrest in S phase. Nucleolin-over-expressed HUVEC were treated with the indicated concentrations of ADP for 72 h. Medium was replaced with fresh medium containing ADP every day. The cell cycles were measured by PI staining. Mock-transfected HUVEC were used as controls. * P<0.05 compared with the control groups.
Figure 4.
Nucleolin protein down-regulation is involved in ADP-induced cell apoptosis.
(A) ADP induced cell detachment in HUVEC. Cells were treated with indicated concentrations of ADP for 72 h. Medium was replaced with fresh medium containing ADP every day. The cells in representative fields were photographed. (B) ADP induced apoptosis in HUVEC. Cells were treated as same as (A). Unfixed cells were stained with FITC-annexin V/PI. Cell apoptosis was measured by flow cytometry analysis. (C) ADP induced time-dependent cell apoptosis. HUVEC were cultured with 100 µM ADP for indicated time periods. Medium was replaced with fresh medium containing ADP every day. Cell apoptosis was measured as same as (B). * P<0.05 compared with the control group. (D) MRS2211 (MRS) did not reverse ADP-induced cell death. HUVEC, pre-treated with the indicated concentrations of MRS2211 for 30 min, were re-treated with 100 µM ADP for 72 h. Cell death was measured by FITC-annexin V/PI staining. * P<0.05 compared with the control group. (E) Nuleolin-over-expression reversed ADP-induced cell death. Nucleolin-over-expressed HUVEC were treated with the indicated concentration of ADP for 72 h. Medium was replaced with fresh medium containing ADP every day. Cell death was measured by FITC-annexin V/PI staining. Mock-transfected HUVEC were used as control. * P<0.05 compared with the control groups.
Figure 5.
Nucleolin protein down-regulation is involved in ADP-induced inhibition of cell proliferation.
(A) Endothelial cell proliferation in response to ADP. HUVEC and HAEC were treated with the indicated concentrations of ADP for 72 h. Medium was replaced with fresh medium containing ADP every day. Cell number was measured with CCK-8 assay. * P<0.05 compared with the control groups. (B) The effect of ADP and UDP on HUVEC proliferation. HUVEC were treated with 100 µM ADP or UDP for the indicated time periods. Medium was replaced with fresh medium containing ADP or UDP every day. Cell number was measured with CCK-8 assay. * P<0.05 compared with the control groups. (C) Endothelial cell proliferation in response to UDP. * P<0.05 compared with the control groups. (D) MRS2211 (MRS) did not reverse ADP-induced inhibition of cell proliferation. HUVEC, pre-treated with the indicated concentrations of MRS2211 for 30 min, were re-treated with ADP for 72 h. Medium was replaced with fresh medium containing MRS2211 and ADP every day. Cell number was measured with CCK-8 assay. * P<0.05 compared with the control groups. (E) MRS2211 (MRS) induced inhibition of cell proliferation. HUVEC were treated with the indicated concentrations of MRS2211 for 72 h. Medium was replaced with fresh medium containing MRS2211 every day. Cell number was measured with CCK-8 assay. * P<0.05 compared with the control group. (F) 2-MeSADP did not induce cell proliferation inhibition. HUVEC were treated with the indicated concentrations of 2-MeSADP for 72 h. Medium was replaced with fresh medium with 2-MeSADP every day. Cell numbers were measured by CCK-8 assay. (G) Nucelolin-over-expression partly reversed ADP-induced inhibition of cell proliferation. Nucleolin-over-expressed HUVEC were treated with the indicated concentrations of nucleolin for 72 h. Medium was replaced with fresh medium containing ADP every day. Mock-treansfected HUVEC were used as control. Cell number was measured with CCK-8 assay. * P<0.05 compared with the control groups.
Figure 6.
ERK pathway is not involved in ADP-induced cell proliferation inhibition.
(A) ADP induced phosphorylation of ERK. HUVEC, starved overnight with serum-free medium, were treated with 100 µM ADP, and lysed at the indicated time points. Western blot was performed for the detection of phospho-ERK1/2 and total ERK1/2 respectively. β-actin protein was detected as a loading control. (B) Quantitation of phosphorylated ERK normalized to total ERK in (A). * P<0.05 compared with the control group. (C) The effect of ERK inhibitor, U0126, on ADP-induced cell proliferation inhibition. HUVEC, pre-treated with the indicated concentrations of U0126 for 30 min, were re-treated with the indicated concentrations of ADP for 24 h, followed with the same treatment once a day in the next 2 days. Cell number was measured by CCK-8 assay. * P<0.05 compared with the non-treated group. # P<0.05 compared with ADP-treated alone group.
Figure 7.
The effect of ADP on cisplatin-induced cell death.
(A) ADP down-regulated the mRNA levels of Bcl-2. HUVEC were treated with indicated concentrations of ADP for 48 h. The mRNA levels of Bcl-2 was detected by RT-PCR. β-actin transcript was detected as loading control. (B, C) ADP down-regulated Bcl-2 protein expression in a dose- (B) and time-dependent (C) manner. (D) The effect of 2-MeSADP on Bcl-2 protein expression. (E) The effect of MRS2211 on Bcl-2 protein expression. (F) The effect of ADP pre-treatment on cisplatin-induced Bcl-2 down-regulation in HUVEC. Cells, pre-treated with the indicated concentrations of ADP for 48 h. were re-treated with 5 µM cisplatin for 48 h. Bcl-2 protein expression was detected by western blot. β-actin protein was detected as a loading control. (G) The effect of ADP pre-treatment on cisplatin-induced Bcl-2 protein expression in HAEC. (H) The effect of ADP pre-treatment on cisplatin-induced cell death. HUVEC were treated as same as (F). The cell survival was detected by CCK-8 assay. * P<0.05 compared with the control group. # P<0.05 compared with cisplatin-treated alone group.
Figure 8.
The effect of ADP on HUVEC cell migration.
(A) HUVEC cells, grown to 100% confluence, were scratched to create a wound and then washed with medium to remove detached cells. Cells were then treated with the indicated concentration of ADP. The wound repairs in representative fields were photographed at time points 0, 12 h to assess the degrees of wound healing. Experiments were performed in triplicate and representative results were shown. (B) The quantitative repair data at time point 6, 12, 24 h.
Figure 9.
The effect of ADP on the proliferation of cervical cancer cells.
(A) ADP down-regulated the protein levels of nucleolin in Caski cervical cancer cells. Cells were treated with 100 µM ADP for the indicated time periods. Nucleolin protein levels were detected by western blot. β-actin protein levels were detected as loading controls. (B) Microscope observation of Caski cells treated with ADP. Cells were treated with 100 µM ADP for 72 h. Cell morphology in the presented field was obtained by microscope. Arrows indicated the detached cell debris. (C) ADP inhibited proliferation of cervical cancer cells. Caski cells were treated with 100 µM ADP for the indicated time periods. Cell numbers were detected every day by CCK-8 assay. P<0.05 compared with the control group. (D) ADP induced cell apoptosis in cervical cancer cells. Caski cells were treated with 100 µM ADP for the indicated time periods. Cell apoptosis was measured by PI/FITC-Annexin V staining assay. P<0.05 compared with the control group. (E) ADP induced cell cycle arrest in S phase. Caski cells were treated with 100 µM ADP for the indicated time periods. Cell cycle was measured by PI staining. P<0.05 compared with the control group.