Journal: Respiratory Research

Article Title: Participation of the PI-3K/Akt-NF-κB signaling pathways in hypoxia-induced mitogenic factor-stimulated Flk-1 expression in endothelial cells

doi: 10.1186/1465-9921-7-101

Figure Lengend Snippet: HIMF induces Flk-1, but not VEGF, expression in mouse endothelial cell line . Endothelial SVEC 4–10 cells were treated with HIMF for various concentrations and periods as indicated. Western blot for VEGF and real-time RT-PCR for Flk-1 expression were performed. (2A) HIMF administration had no impact on VEGF expression in SVEC 4–10 cells. (2B) HIMF induced Flk-1 transcript increase in SVEC 4–10 cells in a dose-dependent manner. Time-course study indicated that HIMF (40 nmol/L)-induced Flk-1 expression can be detected at 6 h, and persisted for 24 h. Triplicate experiments were performed with essentially identical results (n = 3).

Article Snippet: SVEC 4–10, an SV40-transformed murine endothelial cell line [ ], was obtained from the ATCC (CRL-2181) and grown in Dulbecco's Minimal Eagles Medium (DMEM, Gibco Laboratories, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Gibco), penicillin (100 U/ml) and streptomycin (100 μg/ml).

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: Respiratory Research

Article Title: Participation of the PI-3K/Akt-NF-κB signaling pathways in hypoxia-induced mitogenic factor-stimulated Flk-1 expression in endothelial cells

doi: 10.1186/1465-9921-7-101

Figure Lengend Snippet: Generation of HIMF overexpressing endothelial cells . SVEC 4–10 cells were transfected with HIMF cDNA or control vector. Stable cell lines, SVEC-HIMF, along with their transfection control cells SVEC-Zeo, were screened based on resistance to Zeocin (400 μg/ml). Western blots with cell culture medium for HIMF and protein from cell lysate for VEGF (3A), immunofluorescence staining for Flk-1 (3B) and real-time RT-PCR with cell total RNA (3C) demonstrated that SVEC-HIMF cells have higher HIMF protein and mRNA levels than their parent (SVEC 4–10) and vector-transfection (SVEC-Zeo) counterparts. The levels of Flk-1, but not VEGF, in SVEC-HIMF were also increased significantly compared with those of their controls. The symbol (*) indicates a significant increase from parent controls ( P < 0.05). Triplicate experiments were performed with essentially identical results (n = 3).

Article Snippet: SVEC 4–10, an SV40-transformed murine endothelial cell line [ ], was obtained from the ATCC (CRL-2181) and grown in Dulbecco's Minimal Eagles Medium (DMEM, Gibco Laboratories, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Gibco), penicillin (100 U/ml) and streptomycin (100 μg/ml).

Techniques: Transfection, Plasmid Preparation, Stable Transfection, Western Blot, Cell Culture, Immunofluorescence, Staining, Quantitative RT-PCR

Journal: Respiratory Research

Article Title: Participation of the PI-3K/Akt-NF-κB signaling pathways in hypoxia-induced mitogenic factor-stimulated Flk-1 expression in endothelial cells

doi: 10.1186/1465-9921-7-101

Figure Lengend Snippet: HIMF increases the transcription activities, but not mRNA stability, of Flk-1 in SVEC 4–10 cells . (4A) SVEC 4–10, SVEC-zeo and SVEC-HIMF cells were co-transfected with pGL-Flk-1 (-258/+299) and pRL-TK. Twenty-four hours later, cells were lysed with passive lysis buffer, and luciferase activity was measured according to the dual-luciferase assay manual. The results indicated that SVEC-HIMF cells have higher Flk-1 transcription activities than those of their controls. (4B) SVEC 4–10 cells were co-transfected with pGL-Flk-1 (-258/+299) and pRL-TK. Twenty-four hours later, the cells were incubated with HIMF protein as indicated. Then, cells were lysed with passive lysis buffer, and luciferase activity was measured according to the dual-luciferase assay manual. The time-course study demonstrated that HIMF (40 nmol/L)-induced Flk-1 transcription is detectable at 6 h, and persisted for 24 h. After incubation with 10–80 nmol/L of HIMF, Flk-1 promoter activities in SVEC 4–10 were enhanced in a dose-dependent manner. (4C) SVEC 4–10 were treated with different concentrations of HIMF and incubated with 5 μg/ml of Actinomycin D for 6, 12 and 24 h. Real-time RT-PCR indicated that HIMF did not prevent Flk-1 degradation when treated with Actinomycin D in SVEC 4–10 cells. The symbol (*) indicates a significant increase from SVEC 4–10 controls without HIMF ( P < 0.05). Triplicate experiments were performed with essentially identical results (n = 3).

Article Snippet: SVEC 4–10, an SV40-transformed murine endothelial cell line [ ], was obtained from the ATCC (CRL-2181) and grown in Dulbecco's Minimal Eagles Medium (DMEM, Gibco Laboratories, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Gibco), penicillin (100 U/ml) and streptomycin (100 μg/ml).

Techniques: Transfection, Lysis, Luciferase, Activity Assay, Incubation, Quantitative RT-PCR

Journal: Respiratory Research

Article Title: Participation of the PI-3K/Akt-NF-κB signaling pathways in hypoxia-induced mitogenic factor-stimulated Flk-1 expression in endothelial cells

doi: 10.1186/1465-9921-7-101

Figure Lengend Snippet: Promoter deletion assay for HIMF-induced Flk-1 expression in SVEC 4–10 cells . SVEC 4–10 cells were co-transfected with pRL-TK and each Flk-1 luciferase reporter construct (5A) for 24 h, then cells were incubated with HIMF protein (40 nmol/L) for another 24 h. Luciferase activity was measured and the firefly luciferase signal was normalized to the renilla luciferase signal for each individual well. (5B) HIMF induced high Flk-1 promoter activities within cells transfected with pGL-Flk-1 (-258/+299), pGL-Flk-1 (-96/+299) or pGL-Flk-1 (-71/+299), which contain one NF-κB binding site within Flk-1 promoter. Deletion of binding sites for E-Box, Sp1 and AP-2 partially attenuated the transcription activity. In addition, deletion of NF-κB binding site completely abolished HIMF-induced Flk-1 promoter activity. (5C) Further mutation or deletion NF-κB binding site within pGL-Flk-1 (-71/+299) abolished HIMF-induced Flk-1 transcripts in SVEC 4–10 cells. The symbol (*) indicates a significant increase from SVEC 4–10 controls treated without HIMF ( P < 0.05). The symbol (#) indicates a significant decrease from SVEC 4–10 transfected with pGL-Flk-1 (-258/+299) or pGL-Flk-1 (-71/+299) and treated with HIMF ( P < 0.05). Triplicate experiments were performed with essentially identical results (n = 3).

Article Snippet: SVEC 4–10, an SV40-transformed murine endothelial cell line [ ], was obtained from the ATCC (CRL-2181) and grown in Dulbecco's Minimal Eagles Medium (DMEM, Gibco Laboratories, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Gibco), penicillin (100 U/ml) and streptomycin (100 μg/ml).

Techniques: DNA Deletion Assay, Expressing, Transfection, Luciferase, Construct, Incubation, Activity Assay, Binding Assay, Mutagenesis

Journal: Respiratory Research

Article Title: Participation of the PI-3K/Akt-NF-κB signaling pathways in hypoxia-induced mitogenic factor-stimulated Flk-1 expression in endothelial cells

doi: 10.1186/1465-9921-7-101

Figure Lengend Snippet: Activation of NF-κB is essential for HIMF-induced Flk-1 expression . Cells were co-transfected with pNFκB-luc, dominant-negative mutants of NF-κB pathway and pRL-TK, with or without stimulation of HIMF protein for various periods as indicated. (6A) Dual-luciferase assay indicated that SVEC-HIMF had higher NF-κB activity than their control counterparts. (6B) Dual-luciferase assay indicated that HIMF protein increased NF-κB activity in SVEC 4–10 cells in a dose-dependent manner. (6C) Western blots indicated that HIMF (40 nmol/L) induced phosphorylation of IKK and IκBα in SVEC 4–10 cells. Transfection of SVEC 4–10 cells with dominant-negative mutants IKKα (K44A) and IKKβ (K44A), or super-repressor IκBα (S32A/S36A) abolished HIMF (40 nmol/L)-induced NF-κB activity. The figures indicate the relative density compared to control. (6D) The upregulation of Flk-1 induced by HIMF (40 nmol/L) in SVEC 4–10 cells were also attenuated by transfection of these dominant-negative mutants. The symbol (*) indicates a significant increase from SVEC 4–10 parent controls or controls treated without HIMF ( P < 0.05). The symbol (#) indicates a significant decrease from SVEC 4–10 cells treated with HIMF only ( P < 0.05). Triplicate experiments were performed with essentially identical results (n = 3).

Article Snippet: SVEC 4–10, an SV40-transformed murine endothelial cell line [ ], was obtained from the ATCC (CRL-2181) and grown in Dulbecco's Minimal Eagles Medium (DMEM, Gibco Laboratories, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Gibco), penicillin (100 U/ml) and streptomycin (100 μg/ml).

Techniques: Activation Assay, Expressing, Transfection, Dominant Negative Mutation, Luciferase, Activity Assay, Western Blot

Journal: Respiratory Research

Article Title: Participation of the PI-3K/Akt-NF-κB signaling pathways in hypoxia-induced mitogenic factor-stimulated Flk-1 expression in endothelial cells

doi: 10.1186/1465-9921-7-101

Figure Lengend Snippet: HIMF-induced NF-κB activation and upregulation of Flk-1 are PI-3K/Akt pathway dependent . SVEC 4–10 cells were pretreated with signal transduction inhibitors or co-transfected with luciferase constructs and PI-3K dominant-negative mutant, then stimulated with HIMF (40 nmol/L) for various periods as indicated. (7A) HIMF strongly induces phosphorylation of Akt at Ser473 and Thr308. The Akt phosphorylation is detectable at 30 minutes and sustained for 360 min. HIMF also induced phosphorylation of ERK1/2 and p38 MAPK, but not JNKs, in SVEC 4–10 cells. The figures indicate the relative density compared to control. (7B) The PI-3K inhibitor LY294002 (10 μmol/L), but not SB203580 (5 μmol/L), PD098059 (5 μmol/L) or U0126 (5 μmol/L), abolished HIMF-induced Akt phosphorylation and upregulation of Flk-1 in SVEC 4–10 cells. (7C) Transfection of Δp85 into SVEC 4–10 cells abolished HIMF-induced phosphorylation of IKK and IκBα, prevented NF-κB activation and production of Flk-1. The symbol (*) indicates a significant increase from SVEC 4–10 controls without HIMF treatment ( P < 0.05). The symbol (#) indicates a significant decrease from SVEC 4–10 cells treated with HIMF only ( P < 0.05). Triplicate experiments were performed with essentially identical results (n = 3).

Article Snippet: SVEC 4–10, an SV40-transformed murine endothelial cell line [ ], was obtained from the ATCC (CRL-2181) and grown in Dulbecco's Minimal Eagles Medium (DMEM, Gibco Laboratories, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS, Gibco), penicillin (100 U/ml) and streptomycin (100 μg/ml).

Techniques: Activation Assay, Transduction, Transfection, Luciferase, Construct, Dominant Negative Mutation

Journal: Respiratory Research

Article Title: Participation of the PI-3K/Akt-NF-κB signaling pathways in hypoxia-induced mitogenic factor-stimulated Flk-1 expression in endothelial cells

doi: 10.1186/1465-9921-7-101

Figure Lengend Snippet: HIMF induces Flk-1, but not VEGF, expression in mouse endothelial cell line . Endothelial SVEC 4–10 cells were treated with HIMF for various concentrations and periods as indicated. Western blot for VEGF and real-time RT-PCR for Flk-1 expression were performed. (2A) HIMF administration had no impact on VEGF expression in SVEC 4–10 cells. (2B) HIMF induced Flk-1 transcript increase in SVEC 4–10 cells in a dose-dependent manner. Time-course study indicated that HIMF (40 nmol/L)-induced Flk-1 expression can be detected at 6 h, and persisted for 24 h. Triplicate experiments were performed with essentially identical results (n = 3).

Article Snippet: HIMF cDNA or dominant-negative mutants were transfected into SVEC 4–10 cells with Lipofectamine 2000 (Life Technologies, Inc., Gaithersburg, MD).

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: Respiratory Research

Article Title: Participation of the PI-3K/Akt-NF-κB signaling pathways in hypoxia-induced mitogenic factor-stimulated Flk-1 expression in endothelial cells

doi: 10.1186/1465-9921-7-101

Figure Lengend Snippet: Generation of HIMF overexpressing endothelial cells . SVEC 4–10 cells were transfected with HIMF cDNA or control vector. Stable cell lines, SVEC-HIMF, along with their transfection control cells SVEC-Zeo, were screened based on resistance to Zeocin (400 μg/ml). Western blots with cell culture medium for HIMF and protein from cell lysate for VEGF (3A), immunofluorescence staining for Flk-1 (3B) and real-time RT-PCR with cell total RNA (3C) demonstrated that SVEC-HIMF cells have higher HIMF protein and mRNA levels than their parent (SVEC 4–10) and vector-transfection (SVEC-Zeo) counterparts. The levels of Flk-1, but not VEGF, in SVEC-HIMF were also increased significantly compared with those of their controls. The symbol (*) indicates a significant increase from parent controls ( P < 0.05). Triplicate experiments were performed with essentially identical results (n = 3).

Article Snippet: HIMF cDNA or dominant-negative mutants were transfected into SVEC 4–10 cells with Lipofectamine 2000 (Life Technologies, Inc., Gaithersburg, MD).

Techniques: Transfection, Plasmid Preparation, Stable Transfection, Western Blot, Cell Culture, Immunofluorescence, Staining, Quantitative RT-PCR

Journal: Respiratory Research

Article Title: Participation of the PI-3K/Akt-NF-κB signaling pathways in hypoxia-induced mitogenic factor-stimulated Flk-1 expression in endothelial cells

doi: 10.1186/1465-9921-7-101

Figure Lengend Snippet: HIMF increases the transcription activities, but not mRNA stability, of Flk-1 in SVEC 4–10 cells . (4A) SVEC 4–10, SVEC-zeo and SVEC-HIMF cells were co-transfected with pGL-Flk-1 (-258/+299) and pRL-TK. Twenty-four hours later, cells were lysed with passive lysis buffer, and luciferase activity was measured according to the dual-luciferase assay manual. The results indicated that SVEC-HIMF cells have higher Flk-1 transcription activities than those of their controls. (4B) SVEC 4–10 cells were co-transfected with pGL-Flk-1 (-258/+299) and pRL-TK. Twenty-four hours later, the cells were incubated with HIMF protein as indicated. Then, cells were lysed with passive lysis buffer, and luciferase activity was measured according to the dual-luciferase assay manual. The time-course study demonstrated that HIMF (40 nmol/L)-induced Flk-1 transcription is detectable at 6 h, and persisted for 24 h. After incubation with 10–80 nmol/L of HIMF, Flk-1 promoter activities in SVEC 4–10 were enhanced in a dose-dependent manner. (4C) SVEC 4–10 were treated with different concentrations of HIMF and incubated with 5 μg/ml of Actinomycin D for 6, 12 and 24 h. Real-time RT-PCR indicated that HIMF did not prevent Flk-1 degradation when treated with Actinomycin D in SVEC 4–10 cells. The symbol (*) indicates a significant increase from SVEC 4–10 controls without HIMF ( P < 0.05). Triplicate experiments were performed with essentially identical results (n = 3).

Article Snippet: HIMF cDNA or dominant-negative mutants were transfected into SVEC 4–10 cells with Lipofectamine 2000 (Life Technologies, Inc., Gaithersburg, MD).

Techniques: Transfection, Lysis, Luciferase, Activity Assay, Incubation, Quantitative RT-PCR

Journal: Respiratory Research

Article Title: Participation of the PI-3K/Akt-NF-κB signaling pathways in hypoxia-induced mitogenic factor-stimulated Flk-1 expression in endothelial cells

doi: 10.1186/1465-9921-7-101

Figure Lengend Snippet: Promoter deletion assay for HIMF-induced Flk-1 expression in SVEC 4–10 cells . SVEC 4–10 cells were co-transfected with pRL-TK and each Flk-1 luciferase reporter construct (5A) for 24 h, then cells were incubated with HIMF protein (40 nmol/L) for another 24 h. Luciferase activity was measured and the firefly luciferase signal was normalized to the renilla luciferase signal for each individual well. (5B) HIMF induced high Flk-1 promoter activities within cells transfected with pGL-Flk-1 (-258/+299), pGL-Flk-1 (-96/+299) or pGL-Flk-1 (-71/+299), which contain one NF-κB binding site within Flk-1 promoter. Deletion of binding sites for E-Box, Sp1 and AP-2 partially attenuated the transcription activity. In addition, deletion of NF-κB binding site completely abolished HIMF-induced Flk-1 promoter activity. (5C) Further mutation or deletion NF-κB binding site within pGL-Flk-1 (-71/+299) abolished HIMF-induced Flk-1 transcripts in SVEC 4–10 cells. The symbol (*) indicates a significant increase from SVEC 4–10 controls treated without HIMF ( P < 0.05). The symbol (#) indicates a significant decrease from SVEC 4–10 transfected with pGL-Flk-1 (-258/+299) or pGL-Flk-1 (-71/+299) and treated with HIMF ( P < 0.05). Triplicate experiments were performed with essentially identical results (n = 3).

Article Snippet: HIMF cDNA or dominant-negative mutants were transfected into SVEC 4–10 cells with Lipofectamine 2000 (Life Technologies, Inc., Gaithersburg, MD).

Techniques: DNA Deletion Assay, Expressing, Transfection, Luciferase, Construct, Incubation, Activity Assay, Binding Assay, Mutagenesis

Journal: Respiratory Research

Article Title: Participation of the PI-3K/Akt-NF-κB signaling pathways in hypoxia-induced mitogenic factor-stimulated Flk-1 expression in endothelial cells

doi: 10.1186/1465-9921-7-101

Figure Lengend Snippet: Activation of NF-κB is essential for HIMF-induced Flk-1 expression . Cells were co-transfected with pNFκB-luc, dominant-negative mutants of NF-κB pathway and pRL-TK, with or without stimulation of HIMF protein for various periods as indicated. (6A) Dual-luciferase assay indicated that SVEC-HIMF had higher NF-κB activity than their control counterparts. (6B) Dual-luciferase assay indicated that HIMF protein increased NF-κB activity in SVEC 4–10 cells in a dose-dependent manner. (6C) Western blots indicated that HIMF (40 nmol/L) induced phosphorylation of IKK and IκBα in SVEC 4–10 cells. Transfection of SVEC 4–10 cells with dominant-negative mutants IKKα (K44A) and IKKβ (K44A), or super-repressor IκBα (S32A/S36A) abolished HIMF (40 nmol/L)-induced NF-κB activity. The figures indicate the relative density compared to control. (6D) The upregulation of Flk-1 induced by HIMF (40 nmol/L) in SVEC 4–10 cells were also attenuated by transfection of these dominant-negative mutants. The symbol (*) indicates a significant increase from SVEC 4–10 parent controls or controls treated without HIMF ( P < 0.05). The symbol (#) indicates a significant decrease from SVEC 4–10 cells treated with HIMF only ( P < 0.05). Triplicate experiments were performed with essentially identical results (n = 3).

Article Snippet: HIMF cDNA or dominant-negative mutants were transfected into SVEC 4–10 cells with Lipofectamine 2000 (Life Technologies, Inc., Gaithersburg, MD).

Techniques: Activation Assay, Expressing, Transfection, Dominant Negative Mutation, Luciferase, Activity Assay, Western Blot

Journal: Respiratory Research

Article Title: Participation of the PI-3K/Akt-NF-κB signaling pathways in hypoxia-induced mitogenic factor-stimulated Flk-1 expression in endothelial cells

doi: 10.1186/1465-9921-7-101

Figure Lengend Snippet: HIMF-induced NF-κB activation and upregulation of Flk-1 are PI-3K/Akt pathway dependent . SVEC 4–10 cells were pretreated with signal transduction inhibitors or co-transfected with luciferase constructs and PI-3K dominant-negative mutant, then stimulated with HIMF (40 nmol/L) for various periods as indicated. (7A) HIMF strongly induces phosphorylation of Akt at Ser473 and Thr308. The Akt phosphorylation is detectable at 30 minutes and sustained for 360 min. HIMF also induced phosphorylation of ERK1/2 and p38 MAPK, but not JNKs, in SVEC 4–10 cells. The figures indicate the relative density compared to control. (7B) The PI-3K inhibitor LY294002 (10 μmol/L), but not SB203580 (5 μmol/L), PD098059 (5 μmol/L) or U0126 (5 μmol/L), abolished HIMF-induced Akt phosphorylation and upregulation of Flk-1 in SVEC 4–10 cells. (7C) Transfection of Δp85 into SVEC 4–10 cells abolished HIMF-induced phosphorylation of IKK and IκBα, prevented NF-κB activation and production of Flk-1. The symbol (*) indicates a significant increase from SVEC 4–10 controls without HIMF treatment ( P < 0.05). The symbol (#) indicates a significant decrease from SVEC 4–10 cells treated with HIMF only ( P < 0.05). Triplicate experiments were performed with essentially identical results (n = 3).

Article Snippet: HIMF cDNA or dominant-negative mutants were transfected into SVEC 4–10 cells with Lipofectamine 2000 (Life Technologies, Inc., Gaithersburg, MD).

Techniques: Activation Assay, Transduction, Transfection, Luciferase, Construct, Dominant Negative Mutation