cell culture hpaecs  (PromoCell)

Journal: International Journal of Molecular Sciences

Article Title: Combination Therapy with STAT3 Inhibitor Enhances SERCA2a-Induced BMPR2 Expression and Inhibits Pulmonary Arterial Hypertension

doi: 10.3390/ijms22179105

Figure Lengend Snippet: SERCA2a overexpression increases BMPR2 expression in hPAECs. ( A ). SERCA2a (far-red, white color) and BMPR2 (red color) expression was measured by co-immunostaining in hPAECs infected with a control adenovirus encoding β-Galactosidase (Ad.βGal) or SERCA2a (Ad.S2a). Nuclei were counterstained with DAPI (blue). Scale bar: 50 μm. ( B ). BMPR2 mRNA levels were measured by RT-qPCR in hPAECs infected with Ad.S2a or Ad.βGal for 24 hr and were treated with a STAT3 inhibitor (STAT3i; 1 µM) for 48 h, n = 3. ( C ). Representative immunoblots of SERCA2a, BMPR2, phospho-SMAD1-5-9 (p-SMAD-1-5-9), phospho-STAT3 T705 (p-STAT3 T705 ), Total-SMAD1 (T-SMAD1), Total-STAT3 (T-STAT3), and GAPDH in hPAECs in the indicated conditions. ( D – F ). Bar graph represents the quantification of p-STAT3 T705 ( D ), BMPR2 ( E ), p-SMAD1-5-9 ( F ), and normalized to T-STAT3, GAPDH, and T-SMAD1, respectively, n = 3. ( G ). Proliferation levels were measured by BrdU assay in hPAECs overexpressing SERCA2a alone for 48 h and/or were treated with a potent STAT3 inhibitor (STAT3i, 1 µM) for 72 h in a media containing 0.1% or 5% FBS, n = 4. ( H ). SERCA2a mRNA (left) or protein (right) expression was analyzed by RT-qPCR (left) and Western blot (right) in hPAECs overexpressing either a non-silencing shRNA (shNS) or a specific shRNA against SERCA2a (shSERCA2a), n = 3. ( I ). Bar graph represents the quantification of BMPR2 normalized to GAPDH, n = 3. ( J ). BMPR2 mRNA levels were measured by RT-qPCR in hPAECs overexpressing shNS or shSERCA2a alone or in combination with STAT3i (48 h, 1 µM), n = 3. ( K ). STAT3 mRNA or protein expression was analyzed using RT-qPCR (left) and Western blot (right) in hPAECs overexpressing either a non-silencing siRNA (siNS) or a specific siRNA against STAT3 (siSTAT3), n = 3. ( L ). Bar graph represents the quantification of BMPR2 normalized to GAPDH, n = 3. ( M ). BMPR2 mRNA levels were measured by RT-qPCR in hPAECs overexpressing a control vector or SERCA2a alone or in combination with siSTAT3 for 72 h, n = 3. ( N ). Proliferation levels were measured by BrdU assay in hPAECs overexpressing SERCA2a alone and/or BMPR2. Cells were cultured in 0.1% or 5% FBS for 72 h, n = 4. ( O ). Proliferation levels were measured by BrdU assay in hPAECs overexpressing either shSERCA2a alone or with BMPR2. Cells were treated with either 0.1% or 5% FBS for 72 h, n = 4. Data are presented as mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Human pulmonary artery endothelial cells (hPAECs) were purchased from Lonza, Inc. (Allendale, NJ, USA). hPAECs were cultured in EBM-2 medium supplemented with 5% fetal bovine serum (FBS) supplemented with EGM-2 SingleQuots (Lonza). hPAECs were characterized by immunofluorescence with antibodies specific to vWF/Factor VIII and CD31 (PECAM).

Techniques: Over Expression, Expressing, Immunostaining, Infection, Quantitative RT-PCR, Western Blot, BrdU Staining, shRNA, Plasmid Preparation, Cell Culture

Journal: Science signaling

Article Title: The matricellular protein TSP1 promotes human and mouse endothelial cell senescence through CD47 and Nox1

doi: 10.1126/scisignal.aaj1784

Figure Lengend Snippet: Data for human tissue are shown in green; data for cell experiments are shown in red; data in gray are the corresponding controls. (A) Correlation of TSP1 expression as quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR) with age in human lung homogenates (n = 7 subjects; r2=0.69, P = 0.02). Data points are means of three replicates of the same sample. (B) Representative images of TSP1 protein abundance in the intimal and medial layer of human pulmonary arteries of a 73-year-old subject (right; arrows indicate intima) compared to an artery from a 36-year-old subject (left) as determined by immunofluorescence (IF). Scale bar, 100 μm. L, vascular lumen. (C) Quantitation of immunofluorescence images from six subjects (n = 4 samples each, 10 to 20 images per sample) is plotted as a linear regression; equation, r2, and P values are as indicated. MFI, mean fluorescence intensity. (D) Cell cycle profile analysis of vehicle (left) and TSP1-challenged (right) HPAECs measured by propidium iodide (PI) labeling and fluorescence-activated cell sorting (FACS). FL-2, fluorescence channel 2. (E) Quantitative analysis of cell cycle phase distribution of HPAECs. Data are the means ± SEM of 20,000 events (n = 4 biological replicates per treatment group) expressed as percentage. *P < 0.05 for TSP1 challenge compared to vehicle control by Mann-Whitney test of each phase. (F and G) HPAEC proliferation in the presence or absence of TSP1, as measured by trypan blue exclusion assay (F) and MTT assay (G). Data are means ± SEM (n = 3 biological replicates per treatment). P < 0.001 for TSP1 compared to vehicle control by repeated-measures analyses. (H) BrdU incorporation in HPAECs in the presence or absence of TSP1 at 24 hours. Data are means ± SEM (n = 3 biological replicates per treatment). *P < 0.05 for TSP1 challenge compared to vehicle control by Student’s t test.

Article Snippet: HPAEC culture, TSP1 challenge, blocking antibody experiments, and NoxA1ds treatment HPAECs (Lonza) were grown in EBM-2 (endothelial growth basal medium-2) containing EGM-2 (endothelial cell growth medium-2) BulletKit components (Lonza).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Immunofluorescence, Quantitation Assay, Fluorescence, Labeling, FACS, MANN-WHITNEY, Trypan Blue Exclusion Assay, MTT Assay, BrdU Incorporation Assay

Journal: Science signaling

Article Title: The matricellular protein TSP1 promotes human and mouse endothelial cell senescence through CD47 and Nox1

doi: 10.1126/scisignal.aaj1784

Figure Lengend Snippet: Data for TSP1−/− samples are plotted in purple; data for cell experiments are in red; data in gray represent the corresponding controls. (A and B) Abundance of the S phase–associated proliferation marker PCNA in middle-aged TSP1−/− mice (purple bar) compared to age-matched wild-type controls (gray bar) as analyzed by Western blotting (A) and in HPAECs exposed to TSP1 as analyzed by immunofluorescence (B). Data are means ± SEM [n = 6 animals per group (A); n = 3 cell populations per treatment, 10 to 20 images per immunodetection (scale bar, 20 μm)] (B); *P < 0.05 compared to wild-type or vehicle control by Student’s t test. (C to F) Endothelial cell senescence as detected by SA-β-Gal labeling [C; HPAECs and human aortic endothelial cells (HuAoECs)] and increases in cell size (D; HPAECs). SA-β-Gal (E, left) and trichrome staining (E, right) were used on en face tissue to visualize senescence, gross lung vessel morphology, and collagen deposition (blue) in middle-aged wild-type and TSP1−/− lungs. The abundance of SASP factors [MCP-1, p19Arf, and interleukin-6 (IL-6)] was assessed by Western blotting in middle-aged TSP1−/− and age-matched wild-type controls (F; purple compared to gray bars). Data are means ± SEM (n = 3 independent experiments or n = 3 animals; 12 images per group). Scale bar, 100 μm. *P < 0.05 for TSP1 challenge compared to vehicle control or null compared to age-matched wild-type mice by Student’s t test. (G and H) NADPH-driven O2•− (G; cytochrome c assay) and H2O2 (H; Amplex Red assay) production in HPAEC lysates. Data are means ± SEM (n = 4 biological replicates per treatment). *P < 0.05 compared to vehicle controls by Student’s t test. (I and J) En face cell-permeant O2•− scavenger (Tiron)–inhibitable dihydroethidium (DHE)–ROS, as measured by fluorescence microscopy (I), and NADPH-driven homogenate O2•− production, as measured by cytochrome c reduction (J). Data are means ± SEM (n = 3 to 6 mice per group). Scale bar, 50 μm. *P < 0.05 compared to wild-type controls by Student’s t test. a.u., arbitrary units.

Article Snippet: HPAEC culture, TSP1 challenge, blocking antibody experiments, and NoxA1ds treatment HPAECs (Lonza) were grown in EBM-2 (endothelial growth basal medium-2) containing EGM-2 (endothelial cell growth medium-2) BulletKit components (Lonza).

Techniques: Marker, Western Blot, Immunofluorescence, Immunodetection, Labeling, Staining, Amplex Red Assay, Fluorescence, Microscopy

Journal: Science signaling

Article Title: The matricellular protein TSP1 promotes human and mouse endothelial cell senescence through CD47 and Nox1

doi: 10.1126/scisignal.aaj1784

Figure Lengend Snippet: Data points for human tissue are shown in green; data for cell experiments are shown in red; data in gray are the corresponding controls. Data for (A) to (E) are plotted as linear regression (n = 8 samples); equation, r2, and P values are indicated in the corresponding graph. (A and B) Correlation between NADPH-driven O2•− production, as measured by cytochrome c reduction (A), or H2O2 production, as measured by Amplex Red fluorescence (B), and age in human lung homogenates. (C and D) Abundance of Nox1 (C) and p53 and p21cip (D), as measured by Western blot of total homogenates of aging human lung. (E) Intimal immunofluorescence for Nox1 (top, green) and p21cip (bottom, red) in aged human lung sections. Scale bar, 50 μm. Graphs show linear regression analyses. (F) HPAEC senescence induced by TSP1 in the presence or absence of NoxA1ds, as measured by SA-β-Gal staining. Graphical data are means ± SEM (n = 3 biological replicates per treatment). Scale bar, 40 μm. *P < 0.05 for TSP1 challenge compared to scrambled vehicle control by Student’s t test.

Article Snippet: HPAEC culture, TSP1 challenge, blocking antibody experiments, and NoxA1ds treatment HPAECs (Lonza) were grown in EBM-2 (endothelial growth basal medium-2) containing EGM-2 (endothelial cell growth medium-2) BulletKit components (Lonza).

Techniques: Fluorescence, Western Blot, Immunofluorescence, Staining

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