Journal: The Prostate

Article Title: PSMA‐positive membranes secreted from prostate cancer cells have potency to transform vascular endothelial cells into an angiogenic state

doi: 10.1002/pros.24237

Figure Lengend Snippet: PSMA was detected in HUVECs cultured with the conditioned medium derived from LNCaP cells. (A) Confocal images of prostate cancer cells (PC3, DU145, LNCaP cells) cultured in condition 1 or condition 2. The scheme of each condition by which each conditioned medium (CM) was prepared is shown on the right. To prepare the CM, 1.5 × 10 5 cells of prostate cancer cells were seeded on 6‐well plastic dishes (condition 1) or collagen I gels (condition 2). Three days later, the media were replaced with fresh media. The cells were then incubated for another 3 days, then the media were collected as CM. Bars: 100 µm. (B, C) Confocal images of HUVECs cultured with the CM derived from LNCaP cells (B) or PC3 and DU145 cells (C). The CM was diluted to half of its concentration with EBM‐2. The resulting solution was added to the HUVECs seeded on the collagen I gels. Seventy‐two hours later, cells were subjected to immunofluorescence staining for PSMA. Bars: 100 µm. (D) Western blots of HUVEC lysates cultured with the CM derived from prostate cancer cells in condition 2 for 72 h. The CM was diluted to half of its concentration with EBM‐2. The resulting solution was added to the HUVECs seeded on the collagen I gels. The lysates from LNCaP cells were used as a positive control of PSMA expression. (E) The mRNA expression of PSMA in HUVECs cultured with the CM derived from LNCaP cells in condition 2 for 72 h. The CM was diluted to half of its concentration with EBM‐2. The resulting solution was added to the HUVECs seeded on the collagen I gels. Data are mean ± SEM from three independent experiments. ** p < .01. HUVEC, human umbilical vascular endothelial cell; mRNA, messenger RNA; PSMA, prostate‐specific membrane antigen [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Human umbilical vascular endothelial cells (HUVECs) were purchased from Lonza.

Techniques: Cell Culture, Derivative Assay, Incubation, Concentration Assay, Immunofluorescence, Staining, Western Blot, Positive Control, Expressing

Journal: The Prostate

Article Title: PSMA‐positive membranes secreted from prostate cancer cells have potency to transform vascular endothelial cells into an angiogenic state

doi: 10.1002/pros.24237

Figure Lengend Snippet: Fractionation of the CM derived from LNCaP cells. (A) Confocal images of HUVECs cultured with each fraction of CM derived from LNCaP cells. Each fraction was diluted to half of its concentration with EBM‐2 or suspended with EBM‐2, and added to the HUVECs seeded on the collagen I gels. Seventy‐two hours later, cells were subjected to immunofluorescence staining for PSMA. The PSMA‐positive HUVECs were shown by arrowheads. Bars: 100 µm. The representative images from three independent experiments were shown. (B) Confocal images of HUVECs cultured with 10,000 g pellet fractions of CM derived from LNCaP cells. The 10,000 g pellet fraction was labeled with a membrane marker dye, before the suspension in EBM‐2 medium. The labeled 10,000 g pellet was cultured with HUVECs for 6 h, and cells were subjected to immunofluorescence staining for PSMA. Bars: 10 µm (left) and 2 µm (right; magnified images of squares in left images). The representative images from three independent experiments were shown. (C) Western blots of 10,000 g pellet fraction of CM derived from LNCaP cells. The lysates from HUVECs and LNCaP cells were used as negative and positive controls of PSMA expression, respectively. The representative blot data from three independent experiments were shown. (D) Confocal images of HUVECs cultured with the CM derived from PC3 cells that stably express Myc‐PSMA or PSMA‐myc. The CM was diluted in half with EBM‐2, and added to the HUVECs seeded on the collagen I gels. Seventy‐two hours later, cells were subjected to immunofluorescence staining for PSMA. Bars: 100 µm. The representative images from three independent experiments were shown. CM, conditioned medium; HUVEC, human umbilical vascular endothelial cell; mRNA, messenger RNA; PSMA, prostate‐specific membrane antigen [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Human umbilical vascular endothelial cells (HUVECs) were purchased from Lonza.

Techniques: Fractionation, Derivative Assay, Cell Culture, Concentration Assay, Immunofluorescence, Staining, Labeling, Marker, Western Blot, Expressing, Stable Transfection

Journal: The Prostate

Article Title: PSMA‐positive membranes secreted from prostate cancer cells have potency to transform vascular endothelial cells into an angiogenic state

doi: 10.1002/pros.24237

Figure Lengend Snippet: The tube formation assay of HUVECs cultured with 10,000 g pellet fraction of CM derived from prostate cancer cells. (A) Representative images of tube formation. HUVECs seeded on collagen I gel were treated with the 10,000 g pellet fraction of CM derived from LNCaP cells for 6 h, and packed on collagen I followed by VEGF‐A stimulation for 66 h. HUVECs were stained with Calcein‐AM before acquisition of images. To examine the PSMA dependency, the CM was prepared from LNCaP cells depleted of PSMA. Bars: 100 µm. (B) The quantitation of (A). Total tube lengths from three independent experiments were measured and normalized to those of cells cultured with normal EBM‐2. Data are the means ± SEM . * p < .05; n.s., not significant. (C) Western blots of LNCaP cell lysates, 72 h posttransfection with the indicated siRNAs. (D) Confocal images of PC3 cells stably expressing Myc‐PSMA or PSMA‐myc. Bars: 100 µm. (E) Representative images of tube formation. HUVECs seeded on collagen I gel were treated with the 10,000 g pellet fraction of CM derived from PC3 cells for 6 h, and packed in collagen I followed by VEGF‐A stimulation for 66 h. HUVECs were stained with Calcein‐AM before acquisition of images. Bars: 100 µm. (F) The quantitation of (E). Total tube lengths from three independent experiments were measured and normalized to those of PC3 (parental). Data are the means ± SEM . * p < .05; ** p < .01. CM, conditioned medium; HUVEC, human umbilical vascular endothelial cell; mRNA, messenger RNA; PSMA, prostate‐specific membrane antigen [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Human umbilical vascular endothelial cells (HUVECs) were purchased from Lonza.

Techniques: Tube Formation Assay, Cell Culture, Derivative Assay, Staining, Quantitation Assay, Western Blot, Stable Transfection, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Dual Targeting of HIF-1α and DLL4 by Isoxanthohumol Potentiates Immune Checkpoint Blockade

doi: 10.3390/ijms27031576

Figure Lengend Snippet: Screening of natural flavanone derivatives for dual inhibition of HIF-1α and DLL4 promoter activities. ( A , B ) Cytotoxicity assessment of natural flavanone derivatives in HEK293 ( A ) and EA.hy926 ( B ) cells. Cells were treated with 10 µM of each natural flavanone derivative for 24 h, and cell viability was determined by MTT assay. ( C ) Inhibitory effect of flavanone derivatives on HRE-luciferase activity. HEK293 cells were co-transfected with pGL3-HRE and pRL-SV40 Renilla luciferase plasmid for 24 h, followed by treatment with 10 µM of natural flavanone derivatives or STP (used as a positive control) under hypoxic conditions for an additional 24 h. HRE-luciferase activity was measured using a dual-luciferase assay. ( D ) Inhibitory effect of flavanone derivatives on DLL4 promoter activity. EA.hy926 cells were co-transfected with pGL3-DLL4 and pRL-SV40 Renilla luciferase plasmids for 24 h, pre-incubated with 10 µM of natural flavanone derivatives for 1 h, and subsequently incubated for 24 h in the presence or absence of VEGF-A (10 ng/mL). DLL4-luciferase activity was measured using a dual-luciferase assay. Data are presented as the mean ± SD from three independent experiments. ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. control ( A , B ), hypoxic control ( C ), or VEGF-treated control ( D ).

Article Snippet: HEK293 human embryonic kidney epithelial cells (KCLB, Seoul, Republic of Korea), EA.hy926 endothelial cells (ATCC, Manassas, VA, USA), and LLC cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone) and 1% antibiotics (penicillin 100 units/mL and streptomycin 100 mg/mL; Invitrogen, Carlsbad, CA, USA).

Techniques: Inhibition, MTT Assay, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Positive Control, Incubation, Control

Journal: International Journal of Molecular Sciences

Article Title: Dual Targeting of HIF-1α and DLL4 by Isoxanthohumol Potentiates Immune Checkpoint Blockade

doi: 10.3390/ijms27031576

Figure Lengend Snippet: Effect of IXN on HIF-1α and DLL4. ( A , B ) Dual-luciferase assay confirming the inhibitory effects of IXN. Half-maximal inhibitory concentration (IC 50 ) values were calculated using GraphPad Prism software (version 8.0). The red dotted lines indicate the IC 50 values. ( C – F ) HEK293, LLC, and A549 cells were treated with IXN (0–10 µM) under hypoxic conditions for 16 h. HIF-1α protein expression levels were analyzed by Western blot and quantified using ImageJ software (version 1.54p). ( G – I ) EA.hy926 cells were treated with IXN (0–10 µM) in the presence of VEGF-A (10 ng/mL) for 24 h. DLL4 and NICD protein levels were analyzed by Western blot and quantified using ImageJ. Data are presented as the mean ± SD from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 vs. the hypoxic control ( D – F ) or VEGF-treated control ( H , I ).

Article Snippet: HEK293 human embryonic kidney epithelial cells (KCLB, Seoul, Republic of Korea), EA.hy926 endothelial cells (ATCC, Manassas, VA, USA), and LLC cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone) and 1% antibiotics (penicillin 100 units/mL and streptomycin 100 mg/mL; Invitrogen, Carlsbad, CA, USA).

Techniques: Luciferase, Concentration Assay, Software, Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Dual Targeting of HIF-1α and DLL4 by Isoxanthohumol Potentiates Immune Checkpoint Blockade

doi: 10.3390/ijms27031576

Figure Lengend Snippet: Effect of IXN on HIF-1α/VEGF and DLL4/NOTCH1 signaling. ( A ) HEK293 cells were treated with STP (10 µM, positive control) or IXN (10 µM) under hypoxic conditions for 24 h, and HIF-1α protein levels were determined by Western blot. ( B ) EA.hy926 cells were treated with STP (10 µM) or IXN (10 µM) and incubated with or without VEGF-A (10 ng/mL) for 24 h. DLL4 expression was assessed by Western blotting. ( C ) HEK293 cells were treated with STP (10 µM) or IXN (10 µM) under hypoxia for 24 h, and VEGFA mRNA expression was quantified by RT-qPCR. ( D ) EA.hy926 cells were treated with STP (10 µM) or IXN (10 µM) and incubated with or without VEGF-A (10 ng/mL) for 24 h. DLL4 mRNA expression was quantified by RT-qPCR. ( E ) EA.hy926 cell proliferation was evaluated using a BrdU proliferation assay after treatment with STP (10 µM) or IXN (10 µM) in the presence or absence of VEGF-A (10 ng/mL) for 24 h. ( F , G ) Wound healing assay: EA.hy926 monolayers were scratched, treated with STP (10 µM) or IXN (10 µM) for 1 h, followed by VEGF-A (10 ng/mL) stimulation for 24 h. Representative images ( F ) and quantitative analysis of wound closure ( G ) are shown. Yellow dashed lines indicate the wound edges at 0 h (upper line) and 24 h (lower line). Scale bar: 100 µm. ( H – J ) Tube formation assay: EA.hy926 cells were seeded on Matrigel-coated wells with VEGF-A (10 ng/mL) and treated with STP (10 µM) or IXN (10 µM) for 24 h. Representative images were acquired using a light microscope ( H ). Quantitative analysis of total tube length ( I ) and branching points ( J ) was performed using ImageJ software (version 1.54p). Scale bar: 100 µm. ( K , L ) Wound healing assay: EA.hy926 cells were scratched and incubated with either fresh medium or A549-derived conditioned medium (CM) for 24 h. Representative images ( K ) and quantification of wound closure ( L ) are shown. Yellow dashed lines indicate the wound edges at 0 h (upper line) and 24 h (lower line). Scale bar: 100 μm. ( M – O ) Tube formation assay: EA.hy926 cells were seeded on Matrigel and incubated with fresh medium or CM for 8 h. Representative images ( M ) and quantification of total tube length ( N ) and branching points ( O ) are shown. Scale bar: 100 μm. CM were obtained from A549 cells cultured under hypoxic conditions (1% O 2 ) with vehicle (DMSO, CM-Vehicle), STP (10 μM, CM-STP), or IXN (10 μM, CM-IXN) for 24 h. Data are presented as the mean ± SD from three independent experiments. *** p < 0.001, and **** p < 0.0001 vs. hypoxic control ( C ), VEGF-treated control ( D , E , G , I , J ), or CM-Vehicle ( L , N , O ).

Article Snippet: HEK293 human embryonic kidney epithelial cells (KCLB, Seoul, Republic of Korea), EA.hy926 endothelial cells (ATCC, Manassas, VA, USA), and LLC cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone) and 1% antibiotics (penicillin 100 units/mL and streptomycin 100 mg/mL; Invitrogen, Carlsbad, CA, USA).

Techniques: Positive Control, Western Blot, Incubation, Expressing, Quantitative RT-PCR, Proliferation Assay, Wound Healing Assay, Tube Formation Assay, Light Microscopy, Software, Derivative Assay, Cell Culture, Control

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: Poly(I-C) induces endothelial cell apoptosis. A, poly(I-C) induced the detachment of primary HUVECs. Cells, pretreated with 1 μg/ml poly(I-C) for 24 h, were re-stimulated with or without 10 μg/ml poly(I-C) for 24 h, and the cells in representative fields were photographed. Cells, treated with 2 μm staurosporine, were photographed as a positive control. B, poly(I-C) induced apoptosis in primary HUVECs. Cells, pretreated with 1 μg/ml poly(I-C) for 24 h, were re-stimulated with the indicated concentrations of poly(I-C) for 24 h. Unfixed cells were stained with FITC-annexin V/PI. Cell apoptosis was measured by flow cytometry analysis. C, poly(I-C) induced the dose-dependent cell apoptosis in immortalized HUVECs. Cells were treated with the indicated concentrations of poly(I-C) for 24 h. Cell apoptosis was measured as in B. Data are shown as percentages and represented as mean ± S.D. of triplicates. Cells treated with 1 μm staurosporine were used as a positive control. *, p < 0.05 compared with the control group. D, poly(I-C) (10 μg/ml) induced time-dependent cell apoptosis in immortalized HUVECs. *, p < 0.05 compared with the control group.

Article Snippet: Cell Lines and Reagents The immortalized human umbilical vein endothelial cells (HUVECs) expressing endothelial cell characteristic markers, endothelial nitric-oxide synthase, CD31, and Ve-cadherin ( 21 ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Positive Control, Staining, Flow Cytometry, Control

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: Poly(I-C) induces TLR3-dependent apoptosis. A, poly(I-C) up-regulated gene expression of TLR3. Immortalized HUVECs, treated with the indicated concentrations of poly(I-C) for 24 h, were harvested, and the gene expression of TLR3 was measured by RT-PCR. GAPDH transcript was measured as a loading control. B, immortalized HUVECs expressed TLR3 protein. Cells, cultured in the presence or absence of 10 μg/ml poly(I-C) for 24 h, were harvested. The expression of TLR3 protein was measured by flow cytometry. Mouse isotype Ig was used as a control. C, poly(I-C) up-regulated the expression of TLR3 protein in primary HUVECs. Cells, treated with the indicated concentrations of poly(I-C) for 24 h, were harvested and lysed with sample buffer. Equal amounts of total proteins were electrophoresed and blotted for the detection of TLR3 protein expression. β-Actin protein was detected as a loading control. D, poly(I-C) significantly up-regulated the gene expression of TLR3 in primary HUVECs. Cells were treated as in Fig. 1B. The gene transcript of TLR3 was measured by qRT-PCR. GAPDH gene expression was detected as an endogenous control. *, p < 0.05 compared with the control group. E, poly(I-C) induced the phosphorylation of the NF-κB p65 subunit. Immortalized HUVECs, starved overnight with serum-free medium, were cultured in the presence of 2 μg/ml poly(I-C) at 37 °C and lysed at the indicated time points. Phospho-p65 (P-p65) and total p65 (T-p65) were detected by Western blot. F, RT-PCR results show that poly(I-C) up-regulated the gene expression of cytokines in immortalized HUVECs. G, TLR3 neutralization abrogated poly(I-C)-induced cell apoptosis. Immortalized HUVECs (iHUVEC) and 1 μg/ml poly(I-C) pretreated (37 °C for 24 h) primary HUVECs (pHUVEC) were cultured in the absence or presence of mouse anti-human TLR3 antibody at 37 °C for 1 h and then treated with or without 2 μg/ml poly(I-C) for 24 h. Cell apoptosis was tested as in Fig. 1B. Mouse isotype Ig was used as a control. Data are represented as mean ± S.D. of triplicates. *, p < 0.05 compared with the poly(I-C) treatment group. H, TLR3 down-regulation by RNA interference abrogated poly(I-C)-induced cell apoptosis in immortalized HUVECs. Wild-type (Wt), nonspecific shRNA (NSsi), and TLR3 shRNA (TLR3si)-transfected cells were treated with 2 μg/ml poly(I-C) for 24 h. Cell apoptosis was detected as in Fig. 1B. *, p < 0.05 compared with the control group.

Article Snippet: Cell Lines and Reagents The immortalized human umbilical vein endothelial cells (HUVECs) expressing endothelial cell characteristic markers, endothelial nitric-oxide synthase, CD31, and Ve-cadherin ( 21 ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Control, Cell Culture, Expressing, Flow Cytometry, Quantitative RT-PCR, Phospho-proteomics, Western Blot, Neutralization, shRNA, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: Poly(I-C) activates both the intrinsic and extrinsic pathways. A, poly(I-C) activated both caspases 8 and 9. Immortalized HUVECs, treated with the indicated concentrations of poly(I-C) for 24 h, were lysed with sample buffer. The cleaved fragments from caspases 8 and 9 were detected by Western blot. 2 μm staurosporine (SP) was used as a control. B, caspase inhibitors down-regulated the cell apoptosis induced by poly(I-C). Immortalized HUVECs were cultured in the absence or presence of caspase 8 inhibitor (Z-IETD-FMK), caspase 9 inhibitor (Z-LEHD-FMK), or pan-caspase inhibitor (Z-VAD-FMK) at 37 °C for 1 h, followed by the treatment with or without 2 μg/ml poly(I-C) for 24 h. Cell apoptosis was tested as in Fig. 1B. Data are represented as mean ± S.D. of triplicates. *, p < 0.05 compared with the poly(I-C) treatment group. C, Western blot results show that poly(I-C) activated the caspase downstream molecule PARP. D, PARP inhibitors down-regulated the cell apoptosis induced by poly(I-C). *, p < 0.05 compared with the poly(I-C) treatment group.

Article Snippet: Cell Lines and Reagents The immortalized human umbilical vein endothelial cells (HUVECs) expressing endothelial cell characteristic markers, endothelial nitric-oxide synthase, CD31, and Ve-cadherin ( 21 ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Western Blot, Control, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: TNFα and IFNβ do not trigger cell apoptosis. A, poly(I-C) up-regulated the expression of TNFα in immortalized HUVECs. Cells were treated with the indicated concentrations of poly(I-C) for 24 h. The supernatant was harvested to detect the TNFα secretion by ELISA. Data are represented as mean ± S.D. of triplicates. *, p < 0.05 compared with the medium control. B, TNFα (37 °C for 24 h) did not induce cell apoptosis in immortalized HUVECs. C, TNFα (50 ng/ml) induced NF-κB signaling in immortalized HUVECs. D, TNFα neutralization did not inhibit poly(I-C)-induced cell apoptosis in immortalized HUVECs. E, IFNβ (37 °C for 24 h) did not induce cell apoptosis in immortalized HUVECs. F, IFNβ neutralization did not inhibit poly(I-C)-induced cell apoptosis in immortalized HUVECs.

Article Snippet: Cell Lines and Reagents The immortalized human umbilical vein endothelial cells (HUVECs) expressing endothelial cell characteristic markers, endothelial nitric-oxide synthase, CD31, and Ve-cadherin ( 21 ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Control, Neutralization

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: TRAIL-DR4/5 and Noxa trigger the extrinsic and intrinsic pathways, respectively. A and B, RT-PCR results show that poly(I-C) (37 °C for 24 h) up-regulated the gene expression of TRAIL, DR4, and DR5 in immortalized (A) and 1 μg/ml poly(I-C) pretreated primary (B) HUVECs. C, poly(I-C) up-regulated the protein expression of TRAIL in primary HUVECs. Cells, pretreated with 1 μg/ml poly(I-C), were re-treated with the indicated concentrations of poly(I-C) for 24 h. TRAIL in the cell lysates was assayed by ELISA. *, p < 0.05 compared with the control. D, TRAIL neutralization repressed the cell apoptosis induced by poly(I-C) in immortalized HUVECs. *, p < 0.05 compared with the poly(I-C) treatment group. E and F, RT-PCR results show the effect of poly(I-C) (37 °C for 24 h) on the gene expression of Bcl-2 and Noxa in immortalized (E) and primary (F) HUVECs. G and H, Western blot results show the effect of poly(I-C) on the protein expression of Bcl-2 and Noxa in immortalized (G) and primary (H) HUVECs. I, inhibition of TLR3 repressed the poly(I-C)-induced down-regulation of Bcl-2 and up-regulation of Noxa in immortalized HUVECs. Cells were transiently transfected with human TLR3 shRNA plasmid and then treated with 2 μg/ml poly(I-C) for 24 h. The protein expression of TLR3, Bcl-2 and Noxa was detected by Western blot.

Article Snippet: Cell Lines and Reagents The immortalized human umbilical vein endothelial cells (HUVECs) expressing endothelial cell characteristic markers, endothelial nitric-oxide synthase, CD31, and Ve-cadherin ( 21 ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Gene Expression, Expressing, Enzyme-linked Immunosorbent Assay, Control, Neutralization, Western Blot, Inhibition, Transfection, shRNA, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: Roles of p53 and p63 in poly(I-C)-induced cell apoptosis. A, RT-PCR results show that poly(I-C) up-regulated p21 but neither MDM2 nor p53 gene expression. B, quantitative expression of p21, MDM2, and p53 in A by qRT-PCR. *, p < 0.05 compared with the control group. C, effect of p53 inhibitor pifithrin-α (PFT) on poly(I-C)-induced cell apoptosis in immortalized HUVECs. D, p53 expression was down-regulated by RNA interference in immortalized HUVECs. Cells were transiently transfected with p53 siRNA. p53 gene expression was detected 48 h after the transfection. E, effect of p53 down-regulation on poly(I-C)-induced cell apoptosis in immortalized HUVECs. Cells were transiently transfected with p53 siRNA and cultured for 24 h and then were treated with poly(I-C) for 24 h for the detection of cell apoptosis. F and G, poly(I-C) up-regulated the gene expression of TAp63α. Immortalized (F) and 1 μg/ml poly(I-C) pretreated primary (G) HUVECs were treated with the indicated concentrations of poly(I-C) for 24 h. The gene expression of p63 was measured by RT-PCR using a primer pair in the N terminus to detect the transactivation domain and a primer pair in the C terminus to detect p63α, -β, and -γ splices. H and I, Western blot results show that poly(I-C) up-regulated the protein expression of TAp63α in immortalized (H) and primary (I) HUVECs. J, Western blot results show that the TLR3 down-regulation by TLR3 shRNA transient transfection inhibited the expression of TAp63α in primary HUVECs.

Article Snippet: Cell Lines and Reagents The immortalized human umbilical vein endothelial cells (HUVECs) expressing endothelial cell characteristic markers, endothelial nitric-oxide synthase, CD31, and Ve-cadherin ( 21 ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Gene Expression, Expressing, Quantitative RT-PCR, Control, Transfection, Cell Culture, Western Blot, shRNA

Journal: The Journal of Biological Chemistry

Article Title: Toll-like Receptor 3 (TLR3) Induces Apoptosis via Death Receptors and Mitochondria by Up-regulating the Transactivating p63 Isoform α (TAP63α) *

doi: 10.1074/jbc.M110.178798

Figure Lengend Snippet: Modulation of p63 expression represses poly(I-C)-induced cell apoptosis. A, p63 was down-regulated by RNA interference in immortalized HUVECs. Wild-type (Wt), nonspecific shRNA (NSsi), and p63 shRNA (p63si) stably transfected cells were treated with 2 μg/ml poly(I-C) for 24 h. TAp63α protein was measured by Western blot. B, p63 down-regulation repressed poly(I-C)-induced cell apoptosis in immortalized HUVECs. *, p < 0.05 compared with the Wt group. C, Western blot results show that p63 RNA interference inhibited the down-regulation of Bcl-2 and the up-regulation of Noxa in immortalized HUVECs. D, Western blot results show that p63 RNA interference inhibited the activation of caspases 8 and 9 and PARP in immortalized HUVECs. E, RT-PCR results show that p63 RNA interference inhibited the expression of TRAIL, DR4, and DR5 in immortalized HUVECs. F, ΔNp63α was up-regulated by gene transfection in immortalized HUVECs. Cells were transiently transfected with pCDNA3+ (Mock) or pCDNA3+/ΔNp63α and harvested at 48 h post-transfection. The gene expression of ΔNp63α was detected by RT-PCR. G, ΔNp63α overexpression inhibited poly(I-C)-induced cell apoptosis in immortalized HUVECs. Cells were transfected as in F and cultured for 24 h, treated with 2 μg/ml poly(I-C) for another 24 h, and then assayed for cell apoptosis. *, p < 0.05 compared with control groups.

Article Snippet: Cell Lines and Reagents The immortalized human umbilical vein endothelial cells (HUVECs) expressing endothelial cell characteristic markers, endothelial nitric-oxide synthase, CD31, and Ve-cadherin ( 21 ) were obtained from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, shRNA, Stable Transfection, Transfection, Western Blot, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Over Expression, Cell Culture, Control

Journal: Development (Cambridge, England)

Article Title: BRG1 promotes COUP-TFII expression and venous specification during embryonic vascular development

doi: 10.1242/dev.087379

Figure Lengend Snippet: COUP-TFII expression is downregulated in Brg1-deficient endothelial cells. (A-D) Cryosections of littermate control and Brg1fl/fl:Tie2-Cre+ tissues were immunostained for the endothelial cell marker PECAM1 (red), COUP-TFII (green) and the nuclear marker Hoechst (blue). (A,B) Cross-sectioned E10.5 umbilical vessels were immunostained, and although COUP-TFII was expressed in umbilical vein (U.V.) endothelial cells in the control section (A), it was significantly diminished in Brg1fl/fl:Tie2-Cre+ venous endothelial cells (B). U.A., umbilical artery.(C,D) E9.75 embryos were cross-sectioned and immunostained. COUP-TFII was expressed in endothelial cells of the cardinal vein (C.V.) in the control section (C) but was downregulated in Brg1fl/fl:Tie2-Cre+ C.V. endothelial cells (D). D.A., dorsal aorta. For A-D, insets show magnified views of the boxed regions and arrowheads indicate individual endothelial cells. Scale bars: 100 μm. (E,F) Primary endothelial cells (ECs) were isolated from E10.5 control and Brg1fl/fl:Tie2-Cre+ tissues, RNA was purified and cDNA was synthesized. Samples from individual littermate control and Brg1fl/fl:Tie2-Cre+ yolk sacs (E) or embryos (F) were processed for qPCR analysis of Brg1 and COUP-TFII expression. Data from three independent experiments were combined and are presented as relative fold change over the expression levels in control cells±s.em. Significant differences were calculated using a two-tailed Student’s t-test (*P<0.05). (G,H) C166 endothelial cells were transfected with nonspecific (NS) or BRG1-specific siRNA for 24 hours. (G) RNA was isolated, cDNA was synthesized and qPCR for Brg1 or COUP-TFII was performed. Data from three independent experiments were combined and are presented as relative fold change over the expression levels in NS siRNA-treated cells±s.e.m. Significant differences were calculated using a two-tailed Student’s t-test (*P<0.05).(H) Protein samples were subjected to western blot analysis with antibodies that recognize BRG1, COUP-TFII or GAPDH.

Article Snippet: Cell culture and transfections C166 yolk sac endothelial cells (ATCC; CRL-2581) were maintained and transfected with 100 nM BRG1 siGENOME SMART pool or nonspecific control small interfering RNA (siRNA) oligonucleotides (Dharmacon, Lafayette, CO, USA; M-041135-01 and D-001210-01, respectively), as described previously ( Griffin et al., 2011 ).

Techniques: Expressing, Control, Marker, Isolation, Purification, Synthesized, Two Tailed Test, Transfection, Western Blot

Journal: Development (Cambridge, England)

Article Title: BRG1 promotes COUP-TFII expression and venous specification during embryonic vascular development

doi: 10.1242/dev.087379

Figure Lengend Snippet: BRG1 binds to the COUP-TFII promoter in endothelial cells. (A) Alignment of the murine COUP-TFII promoter region with sequences from zebrafish, frog, opossum, dog, chimpanzee and human genomes from the NCBI DCODE website (http://www.dcode.org). Peak heights indicate degree of sequence homology; pink bars above peaks denote evolutionarily conserved regions; yellow represents the COUP-TFII 5′ untranslated region; blue indicates COUP-TFII exon 1. Boxed regions were selected for further analysis of BRG1 binding. Numbers above boxed regions denote approximate distances upstream of the COUP-TFII transcription start site (TSS). (B) Chromatin immunoprecipitation (ChIP) assays were performed on C166 endothelial cells using antibodies against BRG1 or isotype-matched non-specific IgG as a negative control. DNA was isolated and amplified by qPCR to determine whether BRG1 bound to various COUP-TFII promoter regions. Significant BRG1 binding was detected at the -0.3 kb, the -1.2 kb and the -4.7 kb promoter regions. A region upstream of the Fzd5 promoter (Fzd5 UP) was used as a negative control BRG1-binding region, and the Adamts1 promoter served as a positive control BRG1-binding region, as previously described (Griffin et al., 2011). Data from four independent experiments were combined and are presented as fold enrichment over the level of ChIP with negative control IgG antibodies±s.e.m. Significant differences were calculated using a two-tailed Student’s t-test (*P<0.05).

Article Snippet: Cell culture and transfections C166 yolk sac endothelial cells (ATCC; CRL-2581) were maintained and transfected with 100 nM BRG1 siGENOME SMART pool or nonspecific control small interfering RNA (siRNA) oligonucleotides (Dharmacon, Lafayette, CO, USA; M-041135-01 and D-001210-01, respectively), as described previously ( Griffin et al., 2011 ).

Techniques: Sequencing, Binding Assay, Chromatin Immunoprecipitation, Negative Control, Isolation, Amplification, Positive Control, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: BRG1 promotes COUP-TFII expression and venous specification during embryonic vascular development

doi: 10.1242/dev.087379

Figure Lengend Snippet: BRG1 remodels chromatin at the COUP-TFII promoter and influences accessibility of transcriptional machinery. (A,B) C166 endothelial cells were transfected with nonspecific (NS) or BRG1-specific siRNA for 24 hours prior to processing for ChIP assays. (A) ChIP with an antibody against total histone H3 was used to determine nucleosome density at various regions of the COUP-TFII promoter. Nucleosome density was significantly decreased at the -4.7 kb region of the COUP-TFII promoter but was significantly increased at the -1.2 kb and -0.3 kb promoter regions following BRG1 knockdown. (B) ChIP with an antibody against RNA polymerase II (RNAPolII) indicated its ability to bind the -0.3 kb region of the COUP-TFII promoter was significantly decreased following BRG1 knockdown. For A and B, a region upstream of the Fzd5 promoter (Fzd5 UP) was used as a negative control region, and the Adamts1 promoter served as a positive control region for BRG1-induced changes in nucleosome density or RNAPolII binding, respectively. Data from three independent experiments were combined and are presented as fold enrichment over the levels of ChIP with the H3 or RNAPolII antibodies in NS siRNA transfected cells±s.e.m. Significant differences were calculated using a two-tailed Student’s t test (*P<0.05). (C) Model of how BRG1 epigenetically promotes COUP-TFII expression. In wild-type endothelial cells, BRG1 binds the -4.7 kb region of the COUP-TFII promoter, where it mediates chromatin compaction. BRG1 also binds the -1.2 kb and -0.3 kb regions of the promoter, where it mediates chromatin decondensation, thereby allowing binding of RNAPolII close to the COUP-TFII transcription start site. These events promote the expression of COUP-TFII in wild-type cells. By contrast, in Brg1fl/fl:Tie2-Cre+ endothelial cells, which lack BRG1, the -4.7 kb COUP-TFII promoter region undergoes chromatin decondensation, potentially allowing for binding of a transcriptional repressor protein. Likewise, the -1.2 kb and -0.3 kb regions of the promoter undergo chromatin compaction, thereby inhibiting efficient RNAPolII binding and diminishing COUP-TFII expression.

Article Snippet: Cell culture and transfections C166 yolk sac endothelial cells (ATCC; CRL-2581) were maintained and transfected with 100 nM BRG1 siGENOME SMART pool or nonspecific control small interfering RNA (siRNA) oligonucleotides (Dharmacon, Lafayette, CO, USA; M-041135-01 and D-001210-01, respectively), as described previously ( Griffin et al., 2011 ).

Techniques: Transfection, Knockdown, Negative Control, Positive Control, Binding Assay, Two Tailed Test, Expressing

Journal: Development (Cambridge, England)

Article Title: BRG1 promotes COUP-TFII expression and venous specification during embryonic vascular development

doi: 10.1242/dev.087379

Figure Lengend Snippet: BRG1 impacts expression of genes downstream of COUP-TFII signaling. (A) Primary endothelial cells (ECs) were isolated from E10.5 control and Brg1fl/fl:Tie2-Cre+ embryos, RNA was purified, and cDNA was synthesized. Expression levels of Brg1, the arterial markers (red) Nrp1, Hey1, Dll4, Hey2 and Foxc1, and the venous markers (blue) COUP-TFII, Ephb4 and Nrp2 were measured by qPCR. Data from three independent experiments were combined and are presented as relative fold change over the normalized expression level of each gene in control cells (dotted line) ±s.e.m. Significant differences were calculated using a two-tailed Student’s t-test (*P<0.05). (B) C166 cells were transfected with increasing amounts (0.02 ng, 0.2 ng, and 2 ng) of empty vector or comparable amounts of a BRG1 expression plasmid for 24 hours. RNA was isolated, cDNA was synthesized and qPCR for Brg1, COUP-TFII and Ephb4 was performed. Data from three independent experiments were combined and are presented as relative fold change over the normalized expression level of each gene in cells transfected with corresponding amounts of empty vector (dotted line). Bars represent ±s.e.m.; significant differences were calculated using a two-tailed Student’s t-test (*P<0.05). (C) C166 endothelial cells were transfected with nonspecific (NS) siRNA, BRG1-specific siRNA or BRG1 siRNA plus a COUP-TFII expression plasmid for 24 hours. RNA was isolated, cDNA was synthesized and qPCR for Hey2 was performed. Bars represent ±s.e.m. from three independent experiments; significant differences were calculated using a two-tailed Student’s t-test (*P<0.05). (D) Model of how BRG1 impacts venous specification. In arterial endothelial cells (ECs), Notch signaling promotes expression of arterial markers such as Ephrin B2. In venous ECs, BRG1 epigenetically promotes expression of COUP-TFII, presumably in cooperation with an unknown venous-specific co-regulatory protein or transcription factor. COUP-TFII directly inhibits Nrp1 and Foxc1, two upstream mediators of the Notch signaling pathway. COUP-TFII also directly inhibits the downstream Notch effector Hey2 (Chen et al., 2012). As a result of COUP-TFII-mediated Notch pathway inhibition, arterial marker expression is suppressed and the venous marker EPHB4 is expressed.

Article Snippet: Cell culture and transfections C166 yolk sac endothelial cells (ATCC; CRL-2581) were maintained and transfected with 100 nM BRG1 siGENOME SMART pool or nonspecific control small interfering RNA (siRNA) oligonucleotides (Dharmacon, Lafayette, CO, USA; M-041135-01 and D-001210-01, respectively), as described previously ( Griffin et al., 2011 ).

Techniques: Expressing, Isolation, Control, Purification, Synthesized, Two Tailed Test, Transfection, Plasmid Preparation, Inhibition, Marker

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells

doi: 10.1161/ATVBAHA.122.317866

Figure Lengend Snippet: CD70 knockdown reduced cellular NO levels and eNOS (endothelial nitric oxide synthase) expression. Real-time intracellular levels of NO in human aortic endothelial cells (HAECs) and human pulmonary artery endothelial cells (HPAECs) were measured following stimulation with 30 μM ATP. A and B , Show representative averaged time curves for baseline fluorescence, ATP treatment, and washout, with greater fluorescence change (%ΔF intensity ) correlating with greater NO levels. NO levels were reduced following siRNA-mediated CD70 (siCD70) knockdown compared with control-siRNA-treated cells (siCtrl) for HAECs and HPAECs ( C and G ). For HAECs, data represent the median of 89 cells for siCtrl and 129 cells for siCD70, collected over 3 independent experiments. For HPAECs, data represent the median of 105 cells for siCtrl and 106 cells for siCD70 collected over 4 independent experiments. Protein levels ( D and H ) mRNA ( E and I ) for eNOS were decreased after treatment with siCD70 compared with siCtrl. Protein expression of the eNOS chaperone Hsp90 (heat shock protein 90) was reduced with siCD70 treatment in HAECs and HPAECs ( F and J ). For eNOS and Hsp90, representative Western blots are shown along with corresponding densitometry, with values normalized to β-actin levels and expressed as fold change compared with siCtrl. For eNOS mRNA, data are normalized to β-actin mRNA and expressed as fold change compared with siCtrl. Data presented as mean±SE. **** P <0.0001.

Article Snippet: Primary human aortic endothelial cells (HAECs) and pulmonary artery endothelial cells (HPAECs) were purchased from Lonza (Walkersville, MD) and grown in EBM-2 media with EGM-2 supplements (Lonza) but without an antimicrobial additive.

Techniques: Expressing, Fluorescence, Western Blot

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells

doi: 10.1161/ATVBAHA.122.317866

Figure Lengend Snippet: 3-Nitrotyrosine and cellular superoxide are increased and cGMP levels are decreased after CD70 knockdown. Human aortic endothelial cells (HAECs) and human pulmonary artery endothelial cells (HPAECs) were treated with control (siCtrl) vs CD70-directed siRNA (siCD70). Immunoblotting for 3-nitrotyrosine adducts revealed two major species in endothelial cells: a higher molecular weight band corresponding to ≈ 45 kD and a lower molecular weight band corresponding to ≈ 20 kD ( A ). Both bands were increased in siCD70 cells as compared with siCtrl in HAECs ( B and C ) and HPAECs ( D and E ). Total intracellular cGMP levels were likewise decreased in siCD70 cells compared with siCtrl ( F and G ). CD70-knockdown HPAECs loaded with dihydroethidium (DHE) demonstrated increased basal fluorescence in the expected spectrum (excitation 488 nm, emission 588 nm) for 2-hydroethidium, the product of superoxide’s reaction with DHE ( H ). Cells loaded with DHE and then subsequently stimulated with menadione showed increased fluorescence in the setting of CD70 knockdown; furthermore, the reduction in fluorescence with PEG-SOD (pegylated superoxide dismutase) was greater in these cells compared with control cells, supporting the specificity of this increased fluorescence to enhanced superoxide levels. Nitrite/nitrate levels are unchanged in control vs CD70-knockdown cells ( J ). For 3-nitrotyrosine, representative Western blots are shown along with corresponding densitometry, with values normalized to β-actin levels and expressed as fold change compared with siCtrl. Data presented as mean±SE. **** P <0.0001.

Article Snippet: Primary human aortic endothelial cells (HAECs) and pulmonary artery endothelial cells (HPAECs) were purchased from Lonza (Walkersville, MD) and grown in EBM-2 media with EGM-2 supplements (Lonza) but without an antimicrobial additive.

Techniques: Western Blot, Molecular Weight, Fluorescence

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells

doi: 10.1161/ATVBAHA.122.317866

Figure Lengend Snippet: Reduced CD70 expression leads to increased cytosolic and caveolar hydrogen peroxide levels in endothelial cells. Human pulmonary artery endothelial cells (HPAECs) were treated with control (siCtrl) vs CD70-directed siRNA (siCD70) and subsequently transfected with the genetically encoded biosensor Hyper7.2 for real-time intracellular monitoring of hydrogen peroxide (H 2 O 2 ) in the cytosol ( A , B , and E ) and plasmalemmal caveolae ( C , D , and F ). Hydrogen peroxide was assessed by the ratio of fluorescence emission following excitation at 490 nm and 420 nm normalized to baseline fluorescence (normalized R/R 0 ). Following treatment with 1 μM auranofin, cells were treated with 25 μM H 2 O 2 as a positive control. The rate of rise of intracellular H 2 O 2 , calculated as the slope of the linear portion of the auranofin-treatment curve over the final 300 seconds of drug treatment (ΔF intensity /s), was greater in siCD70-treated cells compared with control cells for both cytosolic ( A and B ) and caveolar H 2 O 2 ( C and D ). Treatment with histamine similarly led to greater cytosolic ( E ) and caveolar H 2 O 2 ( F ) levels in CD70-knockdown cells compared with control. All panels were analyzed by unpaired Mann-Whitney U test. *** P <0.001, **** P <0.0001.

Article Snippet: Primary human aortic endothelial cells (HAECs) and pulmonary artery endothelial cells (HPAECs) were purchased from Lonza (Walkersville, MD) and grown in EBM-2 media with EGM-2 supplements (Lonza) but without an antimicrobial additive.

Techniques: Expressing, Transfection, Fluorescence, Positive Control, MANN-WHITNEY

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells

doi: 10.1161/ATVBAHA.122.317866

Figure Lengend Snippet: CD70 knockdown is associated with enhanced NOX (NADPH oxidase) expression. Following CD70 knockdown (siCD70), human pulmonary artery endothelial cells (HPAECs) demonstrated increased NOX (NADPH oxidase) 1 transcript ( A ) and protein ( B ) expression, and NOXA1 mRNA ( C ). Transcript levels of gp91phox, the catalytic subunit of NOX2, were enhanced ( D ), along with the corresponding protein levels of gp91phox ( E ). For NOX1 and gp91phox protein levels, representative Western blots are shown along with corresponding densitometry, with values normalized to β-actin levels and expressed as fold change compared with siCtrl. For NOX1 and NOXA1, mRNA levels are normalized to β-actin mRNA, and for gp91phox, mRNA levels are normalized to RNA polymerase II subunit A mRNA; data are expressed as fold change compared with siCtrl. Data presented as mean±SE.

Article Snippet: Primary human aortic endothelial cells (HAECs) and pulmonary artery endothelial cells (HPAECs) were purchased from Lonza (Walkersville, MD) and grown in EBM-2 media with EGM-2 supplements (Lonza) but without an antimicrobial additive.

Techniques: Expressing, Western Blot

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells

doi: 10.1161/ATVBAHA.122.317866

Figure Lengend Snippet: Reduction in CD70 alters endothelial anti-oxidant enzyme expression. In comparison to control-treated cells (siCtrl), CD70-knockdown cells (siCD70) showed enhanced SOD1 (superoxide dismutase 1) mRNA ( A and H ) and protein ( B and I ) levels in both human aortic endothelial cells (HAECs) and human pulmonary artery endothelial cells (HPAECs). Catalase protein expression was reduced in siCD70 samples ( C and J ); conversely, GPx-1 (glutathione peroxidase 1) mRNA ( D and K ) and protein levels ( E and L ) were elevated. Total intracellular GPx activity was augmented after treatment with siCD70 ( F and M ). Nrf2 (nuclear factor-erythroid factor 2-related factor 2) transcript levels were elevated in siCD70-treated cells ( G and N ). For SOD1, catalase and GPx-1 protein levels, representative Western blots are shown along with corresponding densitometry, with values normalized to β-actin levels and expressed as fold change compared with siCtrl. For SOD1, GPx-1, and Nrf2 mRNA levels, data are normalized to β-actin mRNA and expressed as fold change compared with siCtrl. Data presented as mean±SE.

Article Snippet: Primary human aortic endothelial cells (HAECs) and pulmonary artery endothelial cells (HPAECs) were purchased from Lonza (Walkersville, MD) and grown in EBM-2 media with EGM-2 supplements (Lonza) but without an antimicrobial additive.

Techniques: Expressing, Activity Assay, Western Blot

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Expression of CD70 Modulates Nitric Oxide and Redox Status in Endothelial Cells

doi: 10.1161/ATVBAHA.122.317866

Figure Lengend Snippet: CD70 knockdown leads to increased mitochondrial hydrogen peroxide levels. Following treatment with CD70 siRNA, human pulmonary artery endothelial cells (HPAECs) showed faster accumulation of hydrogen peroxide in the mitochondrial matrix in response to treatment with 1 mmol/L auranofin ( A and B ). SOD2 transcript ( C and E ) and protein ( D and F ) expression were correspondingly increased after CD70 knockdown compared with control cells in human aortic endothelial cells (HAECs) and HPAECs. Representative Western blots are shown for SOD2 along with corresponding densitometry, with values normalized to β-actin levels and expressed as fold change compared with siCtrl. For SOD2 mRNA, data are normalized to β-actin mRNA and expressed as fold change compared with siCtrl. Data presented as mean±SE. **** P <0.0001.

Article Snippet: Primary human aortic endothelial cells (HAECs) and pulmonary artery endothelial cells (HPAECs) were purchased from Lonza (Walkersville, MD) and grown in EBM-2 media with EGM-2 supplements (Lonza) but without an antimicrobial additive.

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: Protein Kinase C beta Mediates CD40 Ligand-Induced Adhesion of Monocytes to Endothelial Cells

doi: 10.1371/journal.pone.0072593

Figure Lengend Snippet: (A) HAECs were incubated with the indicated concentrations of CD40L for 24 h, and static adhesion assays were performed as detailed in Methods. Attached THP-1 cells were visualized and counted on an inverted fluorescent microscopy. Magnification, ×20. (B) Quantification of fluorescence density expressed as means ± SEM. * P< 0.05 vs 0 ng/mL. (C) HAECs were incubated in the presence of (40 ng/mL) for the indicated hours, and then static adhesion assays were performed. * P <0.05 vs 0 h. (D) HAECs were incubated in the presence of PBS (control) or CD40L (80 ng/mL) for 24 h, and static adhesion assays were performed with the use of human peripheral monocytes. * P <0.05 vs control. (D) Platelets were activated as described and incubated with HAECs, then THP-1 cells adhesion was analyzed by static adhesion assays. * P <0.05 vs resting platelets.

Article Snippet: Human aortic endothelial cells (HAECs) were purchased from Cell Applications Inc. (San Diego, CA) and cultured in M199 medium supplemented with FBS (20% vol/vol), penicillin (100 U/mL), streptomycin (100 μg/mL), heparin (90 μg/mL), and endothelial cell growth supplement (20 μg/mL).

Techniques: Incubation, Microscopy, Fluorescence, Control

Journal: PLoS ONE

Article Title: Protein Kinase C beta Mediates CD40 Ligand-Induced Adhesion of Monocytes to Endothelial Cells

doi: 10.1371/journal.pone.0072593

Figure Lengend Snippet: (A and B) HAECs were incubated in the presence of indicated concentrations of for 24 hours. A, Total RNA was isolated and subjected to quantitative RT-PCR to analyze VCAM-1 mRNA levels. * P< 0.05 or ** P< 0.01 vs 0 ng/mL. (B) VCAM-1 protein expression was determined by Western blot. Total cell lysates were subjected to SDS-PAGE and immunoblotting. Blots represent 4 independent experiments with similar results. (D) HAECs were pretreated with antibodies (50 μg/mL) for 30 minutes and then incubated in the presence of PBS (control) or CD40L (80 ng/mL) for 24 h, and static adhesion assays were performed. * P <0.05 vs CD40L. Data are representative of 4 independent experiments with similar results.

Article Snippet: Human aortic endothelial cells (HAECs) were purchased from Cell Applications Inc. (San Diego, CA) and cultured in M199 medium supplemented with FBS (20% vol/vol), penicillin (100 U/mL), streptomycin (100 μg/mL), heparin (90 μg/mL), and endothelial cell growth supplement (20 μg/mL).

Techniques: Incubation, Isolation, Quantitative RT-PCR, Expressing, Western Blot, SDS Page, Control

Journal: PLoS ONE

Article Title: Protein Kinase C beta Mediates CD40 Ligand-Induced Adhesion of Monocytes to Endothelial Cells

doi: 10.1371/journal.pone.0072593

Figure Lengend Snippet: (A and B) HAECs were incubated in the presence of CD40L (80 ng/mL) or PBS (control) for 24 h, and flow adhesion assays were performed at 37°C. In some experiments, HAECs were pretreated with anti–VCAM-1 antibody (50 μg/mL) for 30 minutes before assay. * P <0.05 vs CD40L. Photographs captured from microscope represent 3 independent experiments with similar results. ×20. (B) Quantification of attached THP-1 cells. * P <0.05 vs CD40L. Data are representative of 4 independent experiments with similar results.

Article Snippet: Human aortic endothelial cells (HAECs) were purchased from Cell Applications Inc. (San Diego, CA) and cultured in M199 medium supplemented with FBS (20% vol/vol), penicillin (100 U/mL), streptomycin (100 μg/mL), heparin (90 μg/mL), and endothelial cell growth supplement (20 μg/mL).

Techniques: Incubation, Control, Microscopy

Journal: PLoS ONE

Article Title: Protein Kinase C beta Mediates CD40 Ligand-Induced Adhesion of Monocytes to Endothelial Cells

doi: 10.1371/journal.pone.0072593

Figure Lengend Snippet: HAECs were transfected with vectors encoding the CD40 siRNA or control siRNA and then incubated with CD40L (80 ng/mL) for 24 h. (A) Representative blot showing CD40 protein expression in HAECs transfected with control or CD40 siRNA. (B) VCAM-1 protein expression and (C) THP-1 adhesion were determined as indicated. Data are shown as representative blots or are expressed as the means ± SEM by three independent assays from 4 independent experiments. * P <0.05 vs CD40L plus Control siRNA.

Article Snippet: Human aortic endothelial cells (HAECs) were purchased from Cell Applications Inc. (San Diego, CA) and cultured in M199 medium supplemented with FBS (20% vol/vol), penicillin (100 U/mL), streptomycin (100 μg/mL), heparin (90 μg/mL), and endothelial cell growth supplement (20 μg/mL).

Techniques: Transfection, Control, Incubation, Expressing

Journal: PLoS ONE

Article Title: Protein Kinase C beta Mediates CD40 Ligand-Induced Adhesion of Monocytes to Endothelial Cells

doi: 10.1371/journal.pone.0072593

Figure Lengend Snippet: (A) HAECs were transfected with NF-κB reporter for 24 h and then incubated with the indicated concentrations of CD40L for another 8 h. The luciferase activity was determined using β-gal as the control. Results of three independent experiments are expressed as fold of control. * P <0.05, ** P <0.01 vs 0 ng/mL. (B and C) HAECs were stimulated with the indicated concentrations of CD40L for 2 h. Cells were lysed and the protein extracts were assayed for p65 DNA-binding activity. (C) IκB-α protein expression was measured by immunoblotting. (D) HAECs were infected with Ad-IκB, or Ad-GFP for 24 h and then incubated with CD40L (80 ng/ml) for another 24 h. VCAM-1 protein expression was determined by Western blot. The results were reproducible in 4 independent experiments. * P <0.05.

Article Snippet: Human aortic endothelial cells (HAECs) were purchased from Cell Applications Inc. (San Diego, CA) and cultured in M199 medium supplemented with FBS (20% vol/vol), penicillin (100 U/mL), streptomycin (100 μg/mL), heparin (90 μg/mL), and endothelial cell growth supplement (20 μg/mL).

Techniques: Transfection, Incubation, Luciferase, Activity Assay, Control, Binding Assay, Expressing, Western Blot, Infection

Journal: PLoS ONE

Article Title: Protein Kinase C beta Mediates CD40 Ligand-Induced Adhesion of Monocytes to Endothelial Cells

doi: 10.1371/journal.pone.0072593

Figure Lengend Snippet: (A) HAECs were pretreated with PKCβ inhibitor (5 nM) for 30 minutes and then incubated in the presence of PBS (control) or CD40L (80 ng/mL) for 2 h. Cytosol and nuclear fractions were prepared to measure NF-κB p65 and IκB-α protein expression by immunoblotting. (B) HAECs were pretreated with PKCβ inhibitor (5 nM) for 30 minutes and then incubated in the presence of PBS (control) or CD40L (80 ng/mL) for 24 h. Total cell lysates were prepared and subjected to immunoblotting. Blots represent 4 independent experiments with similar results.

Article Snippet: Human aortic endothelial cells (HAECs) were purchased from Cell Applications Inc. (San Diego, CA) and cultured in M199 medium supplemented with FBS (20% vol/vol), penicillin (100 U/mL), streptomycin (100 μg/mL), heparin (90 μg/mL), and endothelial cell growth supplement (20 μg/mL).

Techniques: Incubation, Control, Expressing, Western Blot