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. 2020 Oct 15;21(20):7622. doi: 10.3390/ijms21207622

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© 2020 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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HPV16 E6 directly interacts with VEGFR-2, but not VEGFR-1. (A) Positive interactions were validated by determining the cell growth on a medium lacking leucine (upper panel) and by the formation of blue colonies (lower panel). β-galactosidase activity (unit), calculated by adding _O-nitrophenyl β-_D-galactopyranoside (ONPG) reagent, is shown below the corresponding lanes. All experiments were repeated at least three times with similar results. (B) Co-immunoprecipitation (Co-IP) of HPV16 E6 with VEGFR-1 or VEGFR-2. Immunoprecipitation (IP) of transfected cells was conducted using anti-Flag antibodies in total lysates, followed by immunoblotting with anti-HPV16 E6, anti-VEGFR-1 and anti-VEGFR-2 antibodies; (left panel) lane 1, pcDNA3.1 (empty-inserted expression vector only) and pcDNA3.1/Flag-HPV16 E6 transfectant; lane 2, pcDNA3.1/Flag-HPV16 E6, and pcDNA3.1/VEGFR-1 transfectant; (right panel) lane 1, pcDNA3.1 (empty expression vector only) and pcDNA3.1/Flag-HPV16 E6 transfectant; lane 2, pcDNA3.1/Flag-HPV16 E6 and pcDNA3.1/VEGFR-2 transfectant. (C) Endogenous proteins in whole cell lysates of human embryonic kidney 293T (HEK293T) cells were introduced into Co-IP with an antibody as indicated, followed by immunoblotting with an anti-HPV16 E6 or anti-VEGFR-2 antibody. Rabbit immunoglobulin G (IgG) and VEGFR-1 served as an IP-negative control. Input (non-IP) immunoblotting data indicated the integrity of cell lysates used for IP. All experiments were repeated at least three times with similar results. (D) HUVECs were treated with VEGF and then control-transfected or transfected with HPV16 E6 or HPV16 E6 plus HPV16 E6-siRNA (siE6#2). Phosphorylation of VEGFR-2 (Tyr-1175) was demonstrated using the specific antibody. The VEGFR-2 band ensured equal loading of samples. All experiments were conducted at least three times with consistent and similar results. (E) Cells were incubated with 10 ng/mL VEGF, and then incubated with either siScramble-transfected or transfected with HPV16 E6 or HPV16 E6 plus HPV16 E6-siRNA (siE6#2). Expression levels of VEGF and HIF-1α protein were determined via immunoblotting analysis. All experiments were repeated at least three times with similar results. (F) Inhibitory effect of VEGFR-2-dependent transcription by HPV16 E6-siRNA (siE6#2). CaSki cervical cells and HUVECs were co-transfected with 500 ng of VEGFR-2-Luc, 500 ng of a VEGFR-2 expression plasmid (pcDNA3.1/VEGFR-2) and increasing concentrations of plasmid-encoding shE6#2 (pcDNA3.1/Flag-siE6#2) (50, 250 and 500 ng). Experiments were conducted in triplicate, and error bars show the mean ± SD; * p < 0.05 compared with control. (G) Suppression of VEGFR-2 kinase activity of HPV16 E6-siRNA (siE6#2) was analysed using an in vitro HTScan VEGFR-2 kinase assay kit combined with colourimetric detection according to the supplier’s instructions. Data are expressed as mean ± SD from three independent experiments; * p < 0.05; ** p < 0.01 compared with zero concentration.

HPV16 E6 directly interacts with VEGFR-2, but not VEGFR-1. (A) Positive interactions were validated by determining the cell growth on a medium lacking leucine (upper panel) and by the formation of blue colonies (lower panel). β-galactosidase activity (unit), calculated by adding _O-nitrophenyl β-_D-galactopyranoside (ONPG) reagent, is shown below the corresponding lanes. All experiments were repeated at least three times with similar results. (B) Co-immunoprecipitation (Co-IP) of HPV16 E6 with VEGFR-1 or VEGFR-2. Immunoprecipitation (IP) of transfected cells was conducted using anti-Flag antibodies in total lysates, followed by immunoblotting with anti-HPV16 E6, anti-VEGFR-1 and anti-VEGFR-2 antibodies; (left panel) lane 1, pcDNA3.1 (empty-inserted expression vector only) and pcDNA3.1/Flag-HPV16 E6 transfectant; lane 2, pcDNA3.1/Flag-HPV16 E6, and pcDNA3.1/VEGFR-1 transfectant; (right panel) lane 1, pcDNA3.1 (empty expression vector only) and pcDNA3.1/Flag-HPV16 E6 transfectant; lane 2, pcDNA3.1/Flag-HPV16 E6 and pcDNA3.1/VEGFR-2 transfectant. (C) Endogenous proteins in whole cell lysates of human embryonic kidney 293T (HEK293T) cells were introduced into Co-IP with an antibody as indicated, followed by immunoblotting with an anti-HPV16 E6 or anti-VEGFR-2 antibody. Rabbit immunoglobulin G (IgG) and VEGFR-1 served as an IP-negative control. Input (non-IP) immunoblotting data indicated the integrity of cell lysates used for IP. All experiments were repeated at least three times with similar results. (D) HUVECs were treated with VEGF and then control-transfected or transfected with HPV16 E6 or HPV16 E6 plus HPV16 E6-siRNA (siE6#2). Phosphorylation of VEGFR-2 (Tyr-1175) was demonstrated using the specific antibody. The VEGFR-2 band ensured equal loading of samples. All experiments were conducted at least three times with consistent and similar results. (E) Cells were incubated with 10 ng/mL VEGF, and then incubated with either siScramble-transfected or transfected with HPV16 E6 or HPV16 E6 plus HPV16 E6-siRNA (siE6#2). Expression levels of VEGF and HIF-1α protein were determined via immunoblotting analysis. All experiments were repeated at least three times with similar results. (F) Inhibitory effect of VEGFR-2-dependent transcription by HPV16 E6-siRNA (siE6#2). CaSki cervical cells and HUVECs were co-transfected with 500 ng of VEGFR-2-Luc, 500 ng of a VEGFR-2 expression plasmid (pcDNA3.1/VEGFR-2) and increasing concentrations of plasmid-encoding shE6#2 (pcDNA3.1/Flag-siE6#2) (50, 250 and 500 ng). Experiments were conducted in triplicate, and error bars show the mean ± SD; * p < 0.05 compared with control. (G) Suppression of VEGFR-2 kinase activity of HPV16 E6-siRNA (siE6#2) was analysed using an in vitro HTScan VEGFR-2 kinase assay kit combined with colourimetric detection according to the supplier’s instructions. Data are expressed as mean ± SD from three independent experiments; * p < 0.05; ** p < 0.01 compared with zero concentration.