*< 0

*< 0.05 versus nontreated control, #< 0.05 less than control antibody treatment with bleomycin but greater than nonCbleomycin-treated control. Requirement of ROS for Bleomycin-Induced PI3K/Akt Activation To determine whether the activation of PI3K/Akt by bleomycin is mediated by ROS, which has not been demonstrated, we studied the effects of bleomycin about cellular ROS generation and PI3K/Akt activation. on fibroblast proliferation and collagen production, suggesting the part of PI3K/Akt in the fibrogenic process. Activation of PI3K/Akt by bleomycin also led to transcriptional activation and protein expression of hypoxia-inducible factor-1 (HIF-1) and vascular endothelial growth factor, which contributed to the fibroproliferative and collagen-inducing effects of bleomycin. The fibrogenic effects of bleomycin were dependent on ROS generation, particularly superoxide anion and hydrogen peroxide, which were induced by bleomycin. Inhibition of ROS generation by antioxidant enzymes, catalase and superoxide dismutase mimetic MnTBAP, abrogated the fibrogenic effects of bleomycin as well as its induction of PI3K/Akt and HIF-1 activation. Together, our results indicate a novel role of PI3K/Akt in fibrogenesis of human lung fibroblasts and its regulation by ROS, which could be exploited for the treatment of pulmonary fibrosis and related disorders. for 5 minutes. Cells were resuspended in 1 ml of Hanks' balanced salt solution and counted using a hemocytometer. A minimum of three separate experiments was performed for each assay. Collagen content was determined by Western blotting as described below and by Sircol assay (Biocolor Ltd, Belfast, UK), according to the manufacturer's protocol. Briefly, Sirius red reagent (50 l) was added to cell culture supernatant (50 l) and mixed for 30 minutes. The collagenCdye complex was precipitated by centrifugation at 16,000 for 5 minutes, washed with ethanol, and dissolved in 0.5 M NaOH. The samples were introduced into a microplate reader and read for ADU-S100 (MIW815) absorbance at 540 nm. Apoptosis Assay Apoptosis was decided using an enzyme-linked immunosorbent assay (ELISA)-based DNA fragmentation assay kit (Roche Molecular Biochem., Indianapolis, IN), according to the manufacturer's instructions. Briefly, cells were lysed with 200 l of lysis buffer at room temperature, and the cell lysate (20 l) was mixed with an antibody solution (80 l) at room temperature for 2 hours. The substrate was then added after the wells were washed three times with a washing buffer. After incubation for 15 minutes at 37C, optical density was measured using a microplate reader at the wavelength of 405 nm. Western Blot Analysis After specific treatments, cells were harvested and lysed on ice for 30 minutes in lysis buffer made up of 150 mM NaCl, 100 mM Tris (pH 8.0), 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, 5 mM EDTA, 10 mM sodium formate, 1 mM sodium orthovanadate, 2 mM leupeptin, 2 mM aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 2 mM pepstatin A. After centrifugation at 14,000 for 15 minutes at 4C, the supernatant was collected as the total cellular protein extract. The protein concentrations were determined using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL). Equal amount of proteins per sample (20 g) were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. The membrane was blocked with T-PBS (0.3% Tween-20 in PBS) containing 3% dry milk and incubated with primary antibody overnight at 4C. After three washes with T-PBS, the membrane was incubated with HRP-conjugated secondary antibody for 1 hour and then washed with 0.05% Tween-20 in PBS. Immunoreactive proteins were detected by chemiluminescence (Supersignal West Pico; Pierce, Rockford, IL) and quantified by imaging densitometry using UN-SCAN-IT digitizing software (Silk Scientific, Orem, UT). Mean densitometry data from impartial experiments were normalized to results in cells from control experiments. ROS Detection Cellular ROS production was decided fluorometrically using dihydroethidine (DHE) and dichlorofluorescein diacetate (DCF-DA) as fluorescent probes for superoxide and peroxide, respectively. After specific treatments, cells were incubated with the probes (10 M) for 30 minutes at 37C, after which they were washed, resuspended in PBS, and analyzed for fluorescence intensity using a multiwell plate reader (FLUOstar OPTIMA; BMG LABTECH Inc., Durham, NC) at the excitation/emission wavelengths of 485/535 nm and 485/610 nm for DHE and DCF fluorescence measurements, respectively. Stable Transfection of Dominant-Negative Akt CRL-1490 cells were cultured in a 6-well plate until they reached 70 to 80% confluence. The cells were transfected with 1 g of CMV-neo vector and 15 l of Lipofectamine 2000 (Invitrogen, Carlsbad, CA) along with 2 g of mutated Akt (SR-Akt T308A/S473A) or control plasmid in the absence of serum. After 5 hours, the medium was replaced with 5% FBS MEM, and 36 hours later they were trypsinized and plated onto 75-ml culture flasks. The cells were then cultured in G418 selection medium (400 g/ml) for 28 days. The selected cells were produced in G418-free MEM for two passages before each experiment. VEGF Protein and Reporter Gene Assays For analysis of VEGF protein, cells were plated in a 6-well plate at a density of 2 105 cells/well in culture medium.Since Akt is activated by bleomycin (Physique 2), we tested whether VEGF could possibly be induced from the bleomycin treatment through Akt signaling. or by dominant-negative Akt (T308A/S473A) manifestation abrogated the consequences of bleomycin on fibroblast proliferation and collagen creation, suggesting the part of PI3K/Akt in the fibrogenic procedure. Activation of PI3K/Akt by bleomycin also resulted in transcriptional activation and proteins manifestation of hypoxia-inducible element-1 (HIF-1) and vascular endothelial development factor, which added towards the fibroproliferative and collagen-inducing ramifications of bleomycin. The fibrogenic ramifications of bleomycin had been reliant on ROS era, especially superoxide anion and hydrogen peroxide, that have been induced by bleomycin. Inhibition of ROS era by antioxidant enzymes, catalase and superoxide dismutase mimetic MnTBAP, abrogated the fibrogenic ramifications of bleomycin aswell as its induction of PI3K/Akt and HIF-1 activation. Collectively, our outcomes indicate a book part of PI3K/Akt in fibrogenesis of human being lung fibroblasts and its own rules by ROS, that could become exploited for the treating pulmonary fibrosis and related disorders. for five minutes. Cells had been resuspended in 1 ml of Hanks' well balanced salt remedy and counted utilizing a hemocytometer. At the least three separate tests was performed for every assay. Collagen content material was dependant on Traditional western blotting as referred to below and by Sircol assay (Biocolor Ltd, Belfast, UK), based on the manufacturer's process. Briefly, Sirius reddish colored reagent (50 l) was put into cell tradition supernatant (50 l) and combined for thirty minutes. The collagenCdye complicated was precipitated by centrifugation at 16,000 for five minutes, cleaned with ethanol, and dissolved in 0.5 M NaOH. The examples had been introduced right into a microplate audience and read for absorbance at 540 nm. Apoptosis Assay Apoptosis was established using an enzyme-linked immunosorbent assay (ELISA)-centered DNA fragmentation assay package (Roche Molecular Biochem., Indianapolis, IN), based on the manufacturer's guidelines. Briefly, cells had been lysed with 200 l of lysis buffer at space temperature, as well as the cell lysate (20 l) was blended with an antibody remedy (80 l) ADU-S100 (MIW815) at space temp for 2 hours. The substrate was after that added following the wells had been cleaned three times having a cleaning buffer. After incubation for quarter-hour at 37C, optical denseness was measured utilizing a microplate audience in the wavelength of 405 nm. Traditional western Blot Evaluation After specific remedies, cells had been gathered and lysed on snow for thirty minutes in lysis buffer including 150 mM NaCl, 100 mM Tris (pH 8.0), 1% Triton X-100, 1% deoxycholic acidity, 0.1% SDS, 5 mM EDTA, 10 mM sodium formate, 1 mM sodium orthovanadate, 2 mM leupeptin, 2 mM aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 2 mM pepstatin A. After centrifugation at 14,000 for quarter-hour at 4C, the supernatant was gathered as the full total mobile protein draw out. The proteins concentrations had been determined utilizing a bicinchoninic acidity protein assay package (Pierce Biotechnology, Rockford, IL). Equivalent quantity of proteins per test (20 g) had been resolved on the 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and moved onto a nitrocellulose membrane. The membrane was clogged with T-PBS (0.3% Tween-20 in PBS) containing 3% dry out milk and incubated with primary antibody overnight at 4C. After three washes with T-PBS, the membrane was incubated with HRP-conjugated supplementary antibody for one hour and then cleaned with 0.05% Tween-20 in PBS. Immunoreactive protein had been recognized by chemiluminescence (Supersignal Western Pico; Pierce, Rockford, IL) and quantified by imaging densitometry using UN-SCAN-IT digitizing software program (Silk Scientific, Orem, UT). Mean densitometry data from 3rd party experiments had been normalized to leads to cells from control tests. ROS Recognition Cellular ROS creation was established fluorometrically using dihydroethidine (DHE) and dichlorofluorescein diacetate (DCF-DA) as fluorescent probes for superoxide and peroxide, respectively. After particular treatments, cells had been incubated using the probes (10 M) for thirty minutes at 37C, and they were cleaned, resuspended in PBS, and examined for fluorescence strength utilizing a multiwell dish audience (FLUOstar OPTIMA; BMG LABTECH Inc., Durham, NC) in the excitation/emission wavelengths of 485/535 nm and 485/610 nm for DHE and DCF fluorescence measurements, respectively. Steady Transfection of Dominant-Negative Akt CRL-1490 cells had been cultured inside a 6-well dish until they reached 70 to 80% confluence. The cells had been transfected with 1 g of CMV-neo vector and 15 l of Lipofectamine 2000 (Invitrogen, Carlsbad, CA) along with 2 g of mutated Akt (SR-Akt T308A/S473A) or control plasmid in the lack of serum. After 5 hours, the moderate was changed with 5% FBS MEM, and 36 hours afterwards these were trypsinized and plated onto 75-ml lifestyle flasks. The cells had been after that cultured in G418 selection moderate (400 g/ml) for 28 times. The chosen cells had been grown up in G418-free of charge MEM for just two passages before every experiment. VEGF Proteins and Reporter Gene Assays For evaluation of VEGF proteins, cells had been plated within a 6-well dish at a thickness of.Furthermore, ROS scavengers, Catalase and MnTBAP, also inhibited bleomycin-induced HIF-1 and VEGF appearance (Statistics 7C and 7D). bleomycin also resulted in transcriptional activation and proteins appearance of hypoxia-inducible aspect-1 (HIF-1) and vascular endothelial development factor, which added towards the fibroproliferative and collagen-inducing ramifications of bleomycin. The fibrogenic ramifications of bleomycin had been reliant on ROS era, especially superoxide anion and hydrogen peroxide, that have been induced by bleomycin. Inhibition of ROS era by antioxidant enzymes, catalase and superoxide dismutase mimetic MnTBAP, abrogated the fibrogenic ramifications of bleomycin aswell as its induction of PI3K/Akt and HIF-1 activation. Jointly, our outcomes indicate a book function of PI3K/Akt in fibrogenesis of individual lung fibroblasts and its own legislation by ROS, that could end up being exploited for the treating pulmonary fibrosis and related disorders. for five minutes. Cells had been resuspended in 1 ml of Hanks' well balanced salt alternative and counted utilizing a hemocytometer. At the least three separate tests was performed for every assay. Collagen articles was dependant on Traditional western blotting as defined below and by Sircol assay (Biocolor Ltd, Belfast, UK), based on the manufacturer's process. Briefly, Sirius crimson reagent (50 l) was put into cell lifestyle supernatant (50 l) and blended for thirty minutes. The collagenCdye complicated was precipitated by centrifugation at 16,000 for five minutes, cleaned with ethanol, and dissolved in 0.5 M NaOH. The examples had been introduced right into a microplate audience and read for absorbance at 540 nm. Apoptosis Assay Apoptosis was driven using an enzyme-linked immunosorbent assay (ELISA)-structured DNA fragmentation assay package (Roche Molecular Biochem., Indianapolis, IN), based on the manufacturer's guidelines. Briefly, cells had been lysed with 200 l of lysis buffer at area temperature, as well as the cell lysate (20 l) was blended with an antibody alternative (80 l) at area heat range for 2 hours. The substrate was after that added following the wells had been cleaned three times using a cleaning buffer. After incubation for a quarter-hour at 37C, optical thickness was measured utilizing a microplate audience on the wavelength of 405 nm. Traditional western Blot Evaluation After specific remedies, cells had been gathered and lysed on glaciers for thirty minutes in lysis buffer filled with 150 mM NaCl, 100 mM Tris (pH 8.0), 1% Triton X-100, 1% deoxycholic acidity, 0.1% SDS, 5 mM EDTA, 10 mM sodium formate, 1 mM sodium orthovanadate, 2 mM leupeptin, 2 mM aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 2 mM pepstatin A. After centrifugation at 14,000 for a quarter-hour at 4C, the supernatant was gathered as the full total mobile protein remove. The proteins concentrations had been determined utilizing a bicinchoninic acidity protein assay package (Pierce Biotechnology, Rockford, IL). Equivalent quantity of proteins per test (20 g) had been resolved on the 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and moved onto a nitrocellulose membrane. The membrane was obstructed with T-PBS (0.3% Tween-20 in PBS) containing 3% dry out milk and incubated with primary antibody overnight at 4C. After three washes with T-PBS, the membrane was incubated with HRP-conjugated supplementary antibody for one hour and then cleaned with 0.05% Tween-20 in PBS. Immunoreactive protein had been discovered by chemiluminescence (Supersignal Western world Pico; Pierce, Rockford, IL) and quantified by imaging densitometry using UN-SCAN-IT digitizing software program (Silk Scientific, Orem, UT). Mean densitometry data from unbiased experiments had been normalized to leads to cells from control tests. ROS Recognition Cellular ROS creation was driven fluorometrically using dihydroethidine (DHE) and dichlorofluorescein diacetate (DCF-DA) as fluorescent probes for superoxide and peroxide, respectively. After particular treatments, cells had been incubated using the probes (10 M) for thirty minutes at 37C, and they were cleaned, resuspended in PBS, and examined for fluorescence strength utilizing a multiwell dish audience (FLUOstar OPTIMA; BMG LABTECH.Comparative fold activity was determined as the normalized reporter activity of the treated sample more than control. Change Transcription PCR Total RNA was extracted with TRIZOL (Invitrogen) and change transcription PCR was performed with Access RT-PCR System (Promega) based on the manufacturer's instructions. aspect-1 (HIF-1) and vascular endothelial development aspect, which contributed towards the fibroproliferative and collagen-inducing ramifications of bleomycin. The fibrogenic ramifications of bleomycin had been reliant on ROS era, especially superoxide anion and hydrogen peroxide, that have been induced by bleomycin. Inhibition of ROS era by antioxidant enzymes, catalase and superoxide dismutase mimetic MnTBAP, abrogated the fibrogenic ramifications of bleomycin aswell as its induction of PI3K/Akt and HIF-1 activation. Jointly, our outcomes indicate a book function of PI3K/Akt in fibrogenesis of individual lung fibroblasts and its own legislation by ROS, that could end up being exploited for the treating pulmonary fibrosis and related disorders. for five minutes. Cells had been resuspended in 1 ml of Hanks' well balanced salt option and counted utilizing a hemocytometer. At the least three separate tests was performed for every assay. Collagen articles was dependant on Traditional western blotting as referred to below and by Sircol assay (Biocolor Ltd, Belfast, UK), based on the manufacturer's process. Briefly, Sirius reddish colored reagent (50 l) was put into cell lifestyle supernatant (50 l) and blended for thirty minutes. The collagenCdye complicated was precipitated by centrifugation at 16,000 for five minutes, cleaned with ethanol, and dissolved in 0.5 M NaOH. The examples had been introduced right into a microplate audience and read for absorbance at 540 nm. Apoptosis Assay Apoptosis was motivated using an enzyme-linked immunosorbent assay (ELISA)-structured DNA fragmentation assay package (Roche Molecular Biochem., Indianapolis, IN), based on the manufacturer's guidelines. Briefly, cells had been lysed with 200 l of lysis buffer at area temperature, as well as the cell lysate (20 l) was blended with an antibody option (80 l) at area temperatures for 2 hours. The substrate was after that added following the wells had been cleaned three times using a cleaning buffer. After incubation for a quarter-hour at 37C, optical thickness was measured utilizing a microplate audience on the wavelength of 405 nm. Traditional western Blot Evaluation After specific remedies, cells had been gathered and lysed on glaciers for thirty minutes in lysis buffer formulated with 150 mM NaCl, 100 mM Tris (pH 8.0), 1% Triton X-100, 1% deoxycholic acidity, 0.1% SDS, 5 mM EDTA, 10 mM sodium formate, 1 mM sodium orthovanadate, 2 mM leupeptin, 2 mM aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 2 mM pepstatin A. After centrifugation at 14,000 for a quarter-hour at 4C, the supernatant was gathered as the full total mobile protein remove. The proteins concentrations had been determined utilizing a bicinchoninic acidity protein assay package (Pierce Biotechnology, Rockford, IL). Equivalent quantity of proteins per test (20 g) had been resolved on the 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and moved onto a nitrocellulose membrane. The membrane was obstructed with T-PBS (0.3% Tween-20 in PBS) containing 3% dry out milk and incubated with primary antibody overnight at 4C. After three washes with T-PBS, the membrane was incubated with HRP-conjugated supplementary antibody for one hour and then cleaned with 0.05% Tween-20 in PBS. Immunoreactive protein had been discovered by chemiluminescence (Supersignal Western world Pico; Pierce, Rockford, IL) and quantified by imaging densitometry using UN-SCAN-IT digitizing software program (Silk Scientific, Orem, UT). Mean densitometry data from indie experiments had been normalized to leads to cells from control tests. ROS Recognition Cellular ROS creation was motivated fluorometrically using dihydroethidine (DHE) and dichlorofluorescein diacetate (DCF-DA) as fluorescent probes for superoxide and peroxide, respectively. After particular treatments, cells had been incubated using the probes (10 M) for thirty minutes at 37C, and they were cleaned, resuspended in PBS, and examined for fluorescence strength utilizing a multiwell dish audience (FLUOstar OPTIMA; BMG LABTECH Inc., Durham, NC) on the excitation/emission wavelengths of 485/535 nm and 485/610 nm for DHE and DCF fluorescence measurements, respectively. Steady Transfection of Dominant-Negative Akt CRL-1490 cells had been cultured within a 6-well dish until they reached 70 to 80% confluence. The cells had been transfected with 1 g of CMV-neo vector and 15 l of Lipofectamine 2000 (Invitrogen, Carlsbad, CA) along with 2 g of mutated Akt (SR-Akt T308A/S473A) or control plasmid in the lack of serum. After 5 hours, the moderate was changed with 5% FBS MEM, and 36 hours.*< 0.05 versus nontreated control, #< 0.05 versus bleomycin-treated control. Since HIF-1 is a known regulator of VEGF (28, 29), we tested whether this transcription aspect is activated with the bleomycin treatment. positions. Inhibition of the phosphorylation by PI3K inhibitors or by dominant-negative Akt (T308A/S473A) appearance abrogated the consequences of bleomycin on fibroblast proliferation and collagen creation, suggesting the function of PI3K/Akt in the fibrogenic procedure. Activation of PI3K/Akt by bleomycin also resulted in transcriptional activation and proteins appearance of hypoxia-inducible aspect-1 (HIF-1) and vascular endothelial development Mouse monoclonal to HSP70 factor, which added towards the fibroproliferative and collagen-inducing effects of bleomycin. The fibrogenic effects of bleomycin were dependent on ROS generation, particularly superoxide anion and hydrogen peroxide, which were induced by bleomycin. Inhibition of ROS generation by antioxidant enzymes, catalase and superoxide dismutase mimetic MnTBAP, abrogated the fibrogenic effects of bleomycin as well as its induction of PI3K/Akt and HIF-1 activation. Together, our results indicate a novel role of PI3K/Akt in fibrogenesis of human lung fibroblasts and its regulation by ROS, which could be exploited for the treatment of pulmonary fibrosis and related disorders. for 5 minutes. Cells were resuspended in 1 ml of Hanks’ balanced salt solution and counted using a hemocytometer. A minimum of three separate experiments was performed for each assay. Collagen content was determined by Western blotting as described below and by Sircol assay (Biocolor Ltd, Belfast, UK), according to the manufacturer’s protocol. Briefly, Sirius red reagent (50 l) was added to cell culture supernatant (50 l) and mixed for 30 minutes. The collagenCdye complex was precipitated by centrifugation at 16,000 for 5 minutes, washed with ethanol, and dissolved in 0.5 M NaOH. The samples were introduced into a microplate reader and read for absorbance at 540 nm. Apoptosis Assay Apoptosis was determined using an enzyme-linked immunosorbent assay (ELISA)-based DNA fragmentation assay kit (Roche Molecular Biochem., Indianapolis, IN), according to the manufacturer’s instructions. Briefly, cells were lysed with 200 l of lysis buffer at room temperature, and the cell lysate (20 l) was mixed with an antibody solution (80 l) at room temperature for 2 hours. The substrate was then added after the wells were washed three times with a washing buffer. After incubation for 15 minutes at 37C, optical density was measured using a microplate reader at the wavelength of 405 nm. Western Blot Analysis After specific treatments, cells were harvested and lysed on ice for 30 minutes in lysis buffer containing 150 mM NaCl, 100 mM Tris (pH 8.0), 1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, 5 mM EDTA, 10 mM sodium formate, 1 mM sodium orthovanadate, 2 mM leupeptin, 2 mM aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, and 2 mM pepstatin A. After centrifugation at 14,000 for 15 minutes at 4C, the supernatant was collected as the total cellular protein extract. The protein concentrations were determined ADU-S100 (MIW815) using a bicinchoninic acid protein assay kit (Pierce Biotechnology, Rockford, IL). Equal amount of proteins per sample (20 g) were resolved on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a nitrocellulose membrane. The membrane was blocked with T-PBS (0.3% Tween-20 in PBS) containing 3% dry milk and incubated with primary antibody overnight at 4C. After three washes with T-PBS, the membrane was incubated with HRP-conjugated secondary antibody for 1 hour and then washed with 0.05% Tween-20 in PBS. Immunoreactive proteins were detected by chemiluminescence (Supersignal West Pico; Pierce, Rockford, IL) and quantified by imaging densitometry using UN-SCAN-IT digitizing software (Silk Scientific, Orem, UT). Mean densitometry data from independent experiments were normalized to results in cells from control experiments. ROS Detection Cellular ROS production was determined fluorometrically using dihydroethidine (DHE) and dichlorofluorescein diacetate (DCF-DA) as fluorescent probes for superoxide and peroxide, respectively. After specific treatments, cells were incubated with the probes (10 M) for 30 minutes at 37C, after which they were washed, resuspended in PBS, and analyzed for fluorescence intensity using a multiwell plate reader (FLUOstar OPTIMA; BMG LABTECH Inc., Durham, NC) at the excitation/emission wavelengths of 485/535 nm and 485/610 nm for DHE and DCF fluorescence measurements, respectively. Stable Transfection of Dominant-Negative Akt CRL-1490 cells were cultured inside a 6-well plate until they reached 70 to 80% confluence. The cells were transfected with 1 g of CMV-neo vector and 15 l of Lipofectamine 2000 (Invitrogen, Carlsbad, CA) along with 2 g of mutated Akt (SR-Akt T308A/S473A) or control plasmid in the absence of serum. After 5 hours, the medium was replaced with 5% FBS MEM, and 36 hours later on they were trypsinized and plated onto 75-ml tradition flasks. The cells were then cultured in G418 selection medium (400 g/ml) for 28 days. The selected cells were cultivated in G418-free MEM for two passages before each experiment. VEGF Protein and Reporter Gene Assays For analysis of VEGF protein, cells were plated in.