Fig 1.
EGFR and VEGFR2 are expressed in THP1 and Raji cell lines.
A total of 106 cells/well were cultured. (A) VEGFR1 and VEGFR3 were found to be absent in both cell lines, using RT-PCR. (B) Cells were shown to express EGFR and VEGFR2 mRNA and (C) proteins using RT-PCR and western blot; respectively. Jurkat cell line was used as a negative control whereas monocytes were used as a positive control.
Fig 2.
EGF activates p-EGFR which induces a significant increase in VEGF.
A total of 106 THP1 or Raji cells/well were cultured in the presence of 20ng/ml EGF at different time intervals. (A) Western blot analysis showing the effect of EGF on the levels of p-EGFR and those of VEGF at different times for THP1 (Left) and Raji (Right) cell lines. (B) ELISA analysis showing the effect of EGF on the levels of secreted VEGF at different times for THP1 (Left) and Raji (Right) cell lines. Results are representatives of four independent experiments (n = 4), for each time point and treatment condition. ELISA results are reported as the mean plus or minus the standard error of the mean (SEM). *, **, *** indicate p<0.05, p<0.001, p<0.0001; respectively.
Fig 3.
Effect of EGFR-induced VEGF on AKT, ERK and p38.
A total of 106 THP1 or Raji cells/well were cultured in the presence of 20ng/ml EGF and the phosphorylation levels of AKT, ERK and p38 were analyzed by Western blot. (A) EGF was treated for the indicated time intervals. Phosphorylation levels of p-AKT, p-ERK and p-p38 increased significantly after EGF stimulation in both THP1 (Left) and Raji (Right) cell lines. (B) In order to measure p-Akt, p-ERK and p-p38, EGFR was blocked using 5μM AG1478 inhibitor for 20min and cells were then stimulated by EGF at 20ng/ml for 10min, 20min, 30min or 5min, 10min, 20min in THP1 or Raji cell lines, respectively. The expression of pAKT, pERK, and p38 was found to be downregulated, using western-blot. It’s worth noting that ERK1/2 and p-ERK1/2 are detected as 2 bands on the western-blot corresponding to ERK1 and ERK2 or their phosphorylated isoforms. Results are representatives of four independent experiments (n = 4), for each time point and treatment condition.
Fig 4.
Effect of EGFR-induced VEGF on phosphorylation of VEGFR2, AKT and ERK.
A total of 106 THP1 or Raji cells/well were cultured in the presence of 20ng/ml EGF. (A) EGF stimulation increased very early the phosphorylation levels of p-VEGFR2 in THP1 (Left) or Raji (Right) cell lines, which peaked at ~5–10 min after treatment. (B) In order to measure p-Akt, p-ERK and p-p38, VEGFR2 was blocked using 20μM DMH4 inhibitor for 1h30min and cells were then stimulated by EGF at 20ng/ml for 10min, 20min, 30min or 5min, 10min, 20min in THP1 or Raji cell lines, respectively. Phosphorylation of AKT and ERK, but not p38, was diminished when the cell lines were pre-incubated with DMH4. (C) Similarly, phosphorylation of AKT and ERK, but not p38, was inhibited when cells were pre-incubated for 1h with 2μg/ml anti-VEGFR2 antibodies. (D) siRNA (20nM) against VEGFR2, left after transfection for 72h, caused a decrease in the phosphorylation levels of AKT and ERK, but not p38. It’s worth noting that ERK1/2 and p-ERK1/2 are detected as 2 bands on the western-blots corresponding to ERK1 and ERK2 or their phosphorylated isoforms. However, when ERK1 antibody is used, only 1 band appears on the blot. Results are representatives of four independent western blot experiments (n = 4).
Fig 5.
Activation of ERK and AKT is PI3K-dependent while PLC regulates p38.
A total of 106 THP1 or Raji cells/well were cultured and pretreated for 30min with 10μM of either LY294002 or U73122 inhibitor. In order to measure p-Akt, p-ERK and p-p38, cells were then treated with EGF at 20ng/ml for 10min, 20min, 30min or 5min, 10min, 20min in THP1 or Raji cell lines, respectively. Western blot analysis showing that EGF-induced phosphorylation of ERK and AKT was inhibited in the presence of LY294002 whereas that of p38 was inhibited by U73122. Results are representatives of three independent experiments (n = 3) for each treatment condition.
Fig 6.
Measurement of intracellular calcium after EGF treatment, in the presence or absence of DMH4.
THP1 or Raji cell lines were cultured as 106 cells/well and incubated, or not, with 20μM DMH4 for lh30min and then treated with 20ng/ml of EGF in a time-course at various time intervals. Fluorometry was then used in order to measure intracellualr calcium concentrations which were significantly and rapidly increased, in both cell lines, but were not affected by VEGFR2 inhibition using 20μM DMH4. Results are representatives of three independent experiments (n = 3), for each time point and treatment condition, reported as the mean plus or minus the standard error of the mean. *, **, *** indicate p<0.05, p<0.001, p<0.0001; respectively.
Fig 7.
Measurement of ROS production after EGF treatment, in the presence or absence of DMH4 or NAC.
THP1 or Raji cell lines were cultured as 106 cells/well and pretreated for 1h30min with DMH4 (1μM or 20μM) or for 30min with N-acetylcysteine (NAC, 5μM) before treatment with 20ng/ml of EGF in a time-course. Following that, H₂O₂ production was measured using fluorometry. (A) Relative fluorescence curves at the basal level, after stimulation with EGF or after inhibition by DMH4 over a period of time. (B) Quantification of the relative fluorescence curve intensities. ROS production was significantly reduced by VEGFR2 inhibition, using 20μM DMH4. (C) In order to measure p-Akt, p-ERK and p-p38, cells were pretreated with 5μM of the ROS inhibitor N-acetylcysteine (NAC) for 30 min and then treated with EGF at 20ng/ml for 10min, 20min, 30min or 5min, 10min, 20min in THP1 or Raji cell lines, respectively. Western blot analysis showed a significant decrease in pAKT, pERK and p-p38. Therefore, VEGFR2 is responsible for EGF-induced ROS production. Results are representatives of three independent experiments (n = 3), reported as the mean plus or minus the standard error of the mean. *, **, *** indicate p<0.05, p<0.001, p<0.0001; respectively.