cell culture hpaecs  (PromoCell)

Journal: Molecular Medicine Reports

Article Title: Effects of NOX1 on fibroblastic changes of endothelial cells in radiation-induced pulmonary fibrosis

doi: 10.3892/mmr.2016.5090

Figure Lengend Snippet: VAS2870 inhibits radiation-induced fibroblastic changes in ECs. (A) HPAECs were irradiated with 5 Gy and incubated for 72 h. VAS2870 (1 µ m) was added to cells 1 h prior to irradiation. To measure ROS, cells were incubated for 30 min with 1 µ m 2′,7′-dichlorodihydrofluorescein diacetate and analyzed by flow cytometry ( * P<0.05 vs. no VAS2870). (B) HPAECs were irradiated with 5 Gy and incubated for 72 h. VAS2870 (1 µ m) was added to cells 1 h prior to irradiation and analysis by immunofluorescence with Alexa 488-conjugated anti-α-SMA and Alexa 594-conjugated anti-CD31 antibodies (green and red, respectively). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue) (scale bars, 20 µ m). (C) Samples were subjected to western blot analysis of α-SMA, vimentin, CD31 and VE-cadherin. β-actin served as the loading control. Protein expression was quantified by densitometric analysis. Values are expressed as the mean ± standard deviation (n=3). * P<0.05 and ** P<0.01 vs. VAS2870-untreated. HPAEC, human pulmonary artery endothelial cell; SMA, smooth-muscle actin; VAS, nicotinamide adenine dinucleotide phosphate oxidase inhibitor VAS2870; ROS, reactive oxygen species; VE, vascular endothelial.

Article Snippet: Endothelial Cell Growth Medium 2 (PromoCell) was used for HPAEC culture.

Techniques: Irradiation, Incubation, Flow Cytometry, Immunofluorescence, Western Blot, Expressing, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: Effects of NOX1 on fibroblastic changes of endothelial cells in radiation-induced pulmonary fibrosis

doi: 10.3892/mmr.2016.5090

Figure Lengend Snippet: NOX1, 2 and 4 shRNAs decrease radiation-induced ROS in HPAECs. (A) Following irradiation, NOX1, 2 and 4 expression was evaluated by RT-qPCR. HPAECs were cultured for the indicated number of days after receiving 5 Gy irradiation. (B) Each lentiviral vector contained a NOX1-, 2- or 4-targeted shRNA, which was then transfected into cultured HPAECs. A lentiviral vector containing a scrambled sequence served as the control. To confirm lentiviral-mediated gene knockdown, NOX1, 2 and 4 expression was analyzed by RT-qPCR. (C) Assessment of ROS and (D) determination of mitochondrial superoxide. Infected cells were irradiated with 5 Gy, followed by incubation with 1 µ m H 2 DCFDA or 2.5 µ m mitoSOX™, respectively, for 30 min and flow cytometric analysis. Values are expressed as the mean ± standard deviation. * P<0.05 (n=6) in C; * P=0.05 (n=3) in D vs. control shRNA. HPAEC, human pulmonary artery endothelial cell; NOX, nicotinamide adenine dinucleotide phosphate oxidase; ROS, reactive oxygen species; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; shRNA, small hairpin RNA; CON, control; RT-qPCR, reverse-transcription quantitative polymerase chain reaction; H 2 DCFDA, 2′,7′-dichlorodihydrofluorescein diacetate.

Article Snippet: Endothelial Cell Growth Medium 2 (PromoCell) was used for HPAEC culture.

Techniques: Irradiation, Expressing, Quantitative RT-PCR, Cell Culture, Plasmid Preparation, shRNA, Transfection, Sequencing, Infection, Incubation, Standard Deviation, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: Effects of NOX1 on fibroblastic changes of endothelial cells in radiation-induced pulmonary fibrosis

doi: 10.3892/mmr.2016.5090

Figure Lengend Snippet: NOX1 shRNA decreases radiation-induced fibrotic changes in HPAECs. (A) Cells transfected with NOX1-, 2- or 4-targeted shRNA were irradiated and incubated for 72 h, followed by western blot analysis of α-SMA, vimentin and CD31. Protein levels were quantified by densitometric analysis of the blots. Values are expressed as the mean ± standard deviation (n=3). **P<0.005; *P<0.05, α-SMA vs. Con (-). (B) HPAECs transfected with NOX1 shRNA were irradiated with 5 Gy and incubated for 72 h. Alexa 488-conjugated anti-FSP1 and Alexa 594-conjugated anti-VE-cadherin antibodies (green and red, respectively) were used to stain cells and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (blue). HPAEC, human pulmonary artery endothelial cell; SMA, smooth-muscle actin; VE, vascular endothelial; NOX, nicotinamide adenine dinucleotide phosphate oxidase; CON, control; shRNA, small hairpin RNA; IR, irradiation; FSP fibroblast-specific protein.

Article Snippet: Endothelial Cell Growth Medium 2 (PromoCell) was used for HPAEC culture.

Techniques: shRNA, Transfection, Irradiation, Incubation, Western Blot, Standard Deviation, Staining