Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Loss of Function in Heparan Sulfate Elongation Genes EXT1 and EXT 2 Results in Improved Nitric Oxide Bioavailability and Endothelial Function

doi: 10.1161/JAHA.114.001274

Figure Lengend Snippet: EXT1 knockdown does not lead to further increase in NOS3 pathway. The effect of EXT1 knockdown on RNA expression of EXT1 (A), EXT2 (B), NOS3 (C), NRF2 (D), KLF2 (E) in HMVEC under laminar flow analyzed by RT‐PCR. EXT1 was silenced using siRNA against EXT1 as described. A scrambled siRNA was used as control. Flow induces a significant increase in NOS3 , nuclear factor erythroid 2?related factor ( NRF2 ) and kruppel like factor 2 ( KLF2 ), which was not further increased by downregulation of EXT1 . Data are from 4 independent experiments and presented as mean±SD. All silencing experiments were performed under laminar flow condition. Differences were analyzed using Student t test. * P <0.05; ** P <0.01. EXT indicates exostosin; HMVEC, human microvascular endothelial cell; KLF2, kruppel like factor 2; NOS, nitric oxide synthesis; NRF‐2, nuclear factor erythroid 2?related factor; RT‐PCR, reverse‐transcription polymerase chain reaction.

Article Snippet: The primary antibody dilutions for Western blots were 1:1000 for rabbit anti‐human phospho‐endothelial nitric oxide synthesis (eNOS) (ser1177, Cell Signalling); 1:1000 for rabbit anti‐human eNOS; 1:1000 for rabbit (Cell Signalling), 1:1000 for rabbit anti‐human AKT2 (Cell Signalling CST‐4691S); and 1:2000 for rabbit anti‐human phospho‐AKT2 Ser473 (Cell Signalling CST‐4060S).

Techniques: Knockdown, RNA Expression, Reverse Transcription Polymerase Chain Reaction, Control, Reverse Transcription, Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Loss of Function in Heparan Sulfate Elongation Genes EXT1 and EXT 2 Results in Improved Nitric Oxide Bioavailability and Endothelial Function

doi: 10.1161/JAHA.114.001274

Figure Lengend Snippet: EXT2 knockdown does not lead to further increase in NOS3 pathway. The effect of EXT2 knockdown on RNA expression of EXT2 (A), EXT2 (B), NOS3 (C), NRF2 (D), KLF2 (E) in HMVEC under laminar flow analyzed by RT‐PCR. EXT1 was silenced using siRNA against EXT2 as described. A scrambled siRNA was used as control. Flow induces a significant increase in NOS3 , NRF2, and KLF2 , which was not further increased by downregulation of EXT2 . Data are from 4 independent experiments and presented as mean±SD. All silencing experiments were performed under laminar flow condition. Differences were analyzed using a nonparametric statistical test (Mann–Whitney). Differences were analyzed using Student t test. * P <0.05; *** P <0.005. EXT indicates exostosin; HMVEC, human microvascular endothelial cell; KLF2, kruppel like factor 2; NOS, nitric oxide synthesis; NRF‐2, nuclear factor erythroid 2?related factor; RT‐PCR, reverse‐transcription polymerase chain reaction.

Article Snippet: The primary antibody dilutions for Western blots were 1:1000 for rabbit anti‐human phospho‐endothelial nitric oxide synthesis (eNOS) (ser1177, Cell Signalling); 1:1000 for rabbit anti‐human eNOS; 1:1000 for rabbit (Cell Signalling), 1:1000 for rabbit anti‐human AKT2 (Cell Signalling CST‐4691S); and 1:2000 for rabbit anti‐human phospho‐AKT2 Ser473 (Cell Signalling CST‐4060S).

Techniques: Knockdown, RNA Expression, Reverse Transcription Polymerase Chain Reaction, Control, MANN-WHITNEY, Reverse Transcription, Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Loss of Function in Heparan Sulfate Elongation Genes EXT1 and EXT 2 Results in Improved Nitric Oxide Bioavailability and Endothelial Function

doi: 10.1161/JAHA.114.001274

Figure Lengend Snippet: Protein expression of eNOS, phospho‐eNOS, AKT and phospho‐AKT2 in HMVEC under static and laminar flow conditions. An example of eNOS, p‐eNOS, (left panel) AKT2 and p‐AKT2 protein expression (right panel) in HMVEC cell lysates under static and flow conditions with or without EXT1 siRNA or EXT2 siRNA . HMVECs were transfected with EXT1 siRNA or EXT2 siRN A , after which cells were studied under laminar flow. β‐Actin was used as loading control. eNOS indicates endothelial nitric oxide synthesis; EXT, exostosin; HMVEC, human microvascular endothelial cell.

Article Snippet: The primary antibody dilutions for Western blots were 1:1000 for rabbit anti‐human phospho‐endothelial nitric oxide synthesis (eNOS) (ser1177, Cell Signalling); 1:1000 for rabbit anti‐human eNOS; 1:1000 for rabbit (Cell Signalling), 1:1000 for rabbit anti‐human AKT2 (Cell Signalling CST‐4691S); and 1:2000 for rabbit anti‐human phospho‐AKT2 Ser473 (Cell Signalling CST‐4060S).

Techniques: Expressing, Transfection, Control

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Loss of Function in Heparan Sulfate Elongation Genes EXT1 and EXT 2 Results in Improved Nitric Oxide Bioavailability and Endothelial Function

doi: 10.1161/JAHA.114.001274

Figure Lengend Snippet: Protein expression of eNOS, phospho‐eNOS, AKT, and phospho‐AKT2 in HMVEC under static and laminar flow conditions. A, The protein expression of eNOS, p‐eNOS, and the ratio of p‐eNOS/eNOS under flow as well as static condition. B, Effect of EXT1 siRNA or EXT2 siRNA on eNOS protein expression. C, Effect of EXT1 siRNA or EXT2 siRNA on phospho‐eNOS protein expression. D, Effect of EXT1 siRNA or EXT2 siRNA on the ratio of phospho‐eNOS/eNOS protein expression. All data are presented as mean±SEM (n=3 experiments). Differences between control and treatment groups were analyzed using nonparametric Kruskal–Wallis statistics. * P <0.05. E, Effect of flow on induction of AKT2 protein expression and phosphorylation status. F, Effect of EXT1 siRNA or EXT2 siRNA on AKT2 protein expression. G, Effect of EXT1 siRNA or EXT2 siRNA on phospho‐AKT2 protein expression. H, Effect of EXT1 siRNA or EXT2 siRNA on the ratio of phospho‐AKT2/AKT2 protein expression. Data are presented as mean±SEM for n=3 experiments. Differences between control and treatment groups were analyzed using nonparametric Kruskal–Wallis statistics. * P <0.05. eNOS indicates endothelial nitric oxide synthesis; EXT, exostosin; HMVEC, human microvascular endothelial cell.

Article Snippet: The primary antibody dilutions for Western blots were 1:1000 for rabbit anti‐human phospho‐endothelial nitric oxide synthesis (eNOS) (ser1177, Cell Signalling); 1:1000 for rabbit anti‐human eNOS; 1:1000 for rabbit (Cell Signalling), 1:1000 for rabbit anti‐human AKT2 (Cell Signalling CST‐4691S); and 1:2000 for rabbit anti‐human phospho‐AKT2 Ser473 (Cell Signalling CST‐4060S).

Techniques: Expressing, Control, Phospho-proteomics

Journal: Molecular medicine reports

Article Title: Upregulation of microRNA‑24 causes vasospasm following subarachnoid hemorrhage by suppressing the expression of endothelial nitric oxide synthase.

doi: 10.3892/mmr.2018.9050

Figure Lengend Snippet: Figure 1. NOS3 is a predicted target gene of miR‑24. The sequences of miR‑24 and the NOS3 3'UTR are presented, revealing the ‘seed sequence’. The potential binding site was mutated using site‑directed mutagenesis to create the mutant NOS3 3'UTR sequence. miR, microRNA; NOS3, endothe lial nitric oxide synthase; UTR, untranslated region.

Article Snippet: Membranes were subsequently incubated with a mouse anti-human NOS3 antibody (1:1,000; cat. no. sc-376751; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or a mouse anti-human β-actin antibody (1:1,000; cat. no. sc-418965; Santa Cruz Biotechnology, Inc.) in 5% non‐fat milk for 16 h at 4 ̊C, following which membranes were washed twice with PBS.

Techniques: Sequencing, Binding Assay, Mutagenesis

Journal: Molecular medicine reports

Article Title: Upregulation of microRNA‑24 causes vasospasm following subarachnoid hemorrhage by suppressing the expression of endothelial nitric oxide synthase.

doi: 10.3892/mmr.2018.9050

Figure Lengend Snippet: Figure 2. NOS3 is a direct target of miR‑24 in VSMCs. A luciferase assay was performed in which wild‑type or mutant NOS3 3'UTR were co‑transfected with an miR‑24 mimic or scramble control. Luciferase activity was measured, and this activity was reduced in VSMCs co‑transfected with wild‑type NOS3 3'UTR and miR‑24, verifying NOS3 as a direct target gene of miR‑24. Data are expressed as the mean ± standard deviation. **P<0.01 vs. scramble control. miR, microRNA; NOS3, endothelial nitric oxide synthase; VSMCs, vascular smooth muscle cells; UTR, untranslated region.

Article Snippet: Membranes were subsequently incubated with a mouse anti-human NOS3 antibody (1:1,000; cat. no. sc-376751; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or a mouse anti-human β-actin antibody (1:1,000; cat. no. sc-418965; Santa Cruz Biotechnology, Inc.) in 5% non‐fat milk for 16 h at 4 ̊C, following which membranes were washed twice with PBS.

Techniques: Luciferase, Mutagenesis, Control, Activity Assay, Standard Deviation

Journal: Molecular medicine reports

Article Title: Upregulation of microRNA‑24 causes vasospasm following subarachnoid hemorrhage by suppressing the expression of endothelial nitric oxide synthase.

doi: 10.3892/mmr.2018.9050

Figure Lengend Snippet: Figure 4. Expression levels of miR‑24 and NOS3 in SAH patients with or without vasospasm. (A) Expression levels of miR‑24 and (B) mRNA and (C) protein expression levels of NOS3 were analyzed in SAH patients with or without vasospasm. miR‑24 expression levels were increased in SAH patients with vasospasm compared with those without, whereas NOS3 expression levels were decreased in SAH patients with vasospasm compared with those without. *P<0.05 vs. SAH with vasospasm. miR, microRNA; NOS3, endothe lial nitric oxide synthase; SAH, subarachnoid hemorrhage.

Article Snippet: Membranes were subsequently incubated with a mouse anti-human NOS3 antibody (1:1,000; cat. no. sc-376751; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or a mouse anti-human β-actin antibody (1:1,000; cat. no. sc-418965; Santa Cruz Biotechnology, Inc.) in 5% non‐fat milk for 16 h at 4 ̊C, following which membranes were washed twice with PBS.

Techniques: Expressing

Journal: Molecular medicine reports

Article Title: Upregulation of microRNA‑24 causes vasospasm following subarachnoid hemorrhage by suppressing the expression of endothelial nitric oxide synthase.

doi: 10.3892/mmr.2018.9050

Figure Lengend Snippet: Figure 3. Negative regulatory association between miR‑24 and NOS3. miR‑24 and NOS3 expression levels were analyzed by reverse transcrip tion‑quantitative polymerase chain reaction in vascular tissues from 29 subarachnoid hemorrhage patients. There was a negative correlation between miR‑24 and NOS3 expression levels. P<0.05. miR, microRNA; NOS3, endo thelial nitric oxide synthase.

Article Snippet: Membranes were subsequently incubated with a mouse anti-human NOS3 antibody (1:1,000; cat. no. sc-376751; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or a mouse anti-human β-actin antibody (1:1,000; cat. no. sc-418965; Santa Cruz Biotechnology, Inc.) in 5% non‐fat milk for 16 h at 4 ̊C, following which membranes were washed twice with PBS.

Techniques: Expressing, Polymerase Chain Reaction

Journal: Molecular medicine reports

Article Title: Upregulation of microRNA‑24 causes vasospasm following subarachnoid hemorrhage by suppressing the expression of endothelial nitric oxide synthase.

doi: 10.3892/mmr.2018.9050

Figure Lengend Snippet: Figure 5. Effect of miR‑24 on the expression of NOS3. VSMCs were transfected with an miR‑24 mimic, NOS3 siRNA, an miR‑24 inhibitor or scramble control, and the NOS3 (A) protein and (B) mRNA expression levels were subsequently assessed by western blotting and reverse transcription‑quantitative polymerase chain reaction, respectively. VSMCs transfected with an miR‑24 mimic or NOS3 siRNA exhibited decreased expression levels of NOS3 compared with the scramble control, whereas transfection with an miR‑24 inhibitor increased NOS3 expression levels. Data are expressed as the mean ± standard deviation. *P<0.05 vs. scramble control. miR, microRNA; NOS3, endothelial nitric oxide synthase; VSMCs, vascular smooth muscle cells; siRNA, small interfering RNA.

Article Snippet: Membranes were subsequently incubated with a mouse anti-human NOS3 antibody (1:1,000; cat. no. sc-376751; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) or a mouse anti-human β-actin antibody (1:1,000; cat. no. sc-418965; Santa Cruz Biotechnology, Inc.) in 5% non‐fat milk for 16 h at 4 ̊C, following which membranes were washed twice with PBS.

Techniques: Expressing, Transfection, Control, Western Blot, Polymerase Chain Reaction, Standard Deviation, Small Interfering RNA

Journal: Endocrinology

Article Title: Phytoestrogen Genistein Up-Regulates Endothelial Nitric Oxide Synthase Expression Via Activation of cAMP Response Element-Binding Protein in Human Aortic Endothelial Cells

doi: 10.1210/en.2012-1076

Figure Lengend Snippet: Genistein (G) enhances eNOS protein expression and NO production in HAEC. Confluent HAEC were incubated with various concentrations (1–10 μ m ) of genistein or vehicle (DMSO) for 5 d. A, eNOS protein levels in cell extracts were measured by Western blotting and normalized to β-actin content. B, Nitrite/nitrate (NO x ) production stimulated by ionophore 23187 was measured using a fluorometric assay kit and normalized to protein content. Values (mean ± se ) of three separate experiments are expressed as fold over vehicle alone-treated cells. A set of representative images and bar graph (mean ± se ) are shown. *, P < 0.05 vs. vehicle alone-treated cells.

Article Snippet: Primary human aortic EC (HAEC) and endothelial growth supplements (EGM2) were purchased from Lonza Walkersville (Walkersville, MD); M199 media and fetal bovine serum (FBS) were obtained from Invitrogen (Carlsbad, CA); pGL2-eNOS promoter-luciferase plasmid (eNOS-Luc) was from Addgene, Inc. (Cambridge, MA); PKA activity and dual luciferase reporter assay kits were from Promega (Madison, WI); CREB ShortCut small interfering RNA (siRNA), scramble sequence of siRNA, and transfection reagents were from New England Biolabs (Ipswich, MA); transfection reagents were obtained from Targeting System (Santee, CA); nitrite/nitrate fluorometric assay reagents were purchased from Cayman Chemical (Ann Arbor, MI); antibodies for eNOS, phospho-CREB, CREB, and β-actin were from Cell Signaling Technology (Beverly, MA); nitrocellulose membranes and protein assay kits were from Bio-Rad (Hercules, CA); genistein, protease, and phosphatase inhibitor cocktails, H89, P3115, PD98059, LY294002, and other general chemicals were from Sigma (St. Louis, MO).

Techniques: Expressing, Incubation, Western Blot

Journal: Endocrinology

Article Title: Phytoestrogen Genistein Up-Regulates Endothelial Nitric Oxide Synthase Expression Via Activation of cAMP Response Element-Binding Protein in Human Aortic Endothelial Cells

doi: 10.1210/en.2012-1076

Figure Lengend Snippet: The stimulatory effect of genistein (G) on NO production is not dependent on the PKC, PI3K, or ERK pathway in HAEC. Confluent HAEC were preincubated with either P3115 (100 μ m ), a PKC inhibitor (A and B), LY294002 (LY) (10 μ m ), the PI3K inhibitor (C and D), or PD 98059 (PD) (10 μ m ), a ERK inhibitor (E and F), for 30 min followed by addition of G (10 μ m ) or vehicle (V) for 5 d. Protein expression of eNOS in cell extracts was analyzed with Western blotting and normalized to β-actin content. Values (mean ± se ) of three separate experiments are expressed as fold of control, and a set of representative images and bar graph (mean ± se ) are shown (A, C, and E). Nitrite/nitrate (NO x ) production stimulated by ionophore 23187 was measured and normalized to protein content (B, D, and F). Values (mean ± se ) of three independent experiments are expressed as fold over control; *, P < 0.05 vs. vehicle alone-treated cells.

Article Snippet: Primary human aortic EC (HAEC) and endothelial growth supplements (EGM2) were purchased from Lonza Walkersville (Walkersville, MD); M199 media and fetal bovine serum (FBS) were obtained from Invitrogen (Carlsbad, CA); pGL2-eNOS promoter-luciferase plasmid (eNOS-Luc) was from Addgene, Inc. (Cambridge, MA); PKA activity and dual luciferase reporter assay kits were from Promega (Madison, WI); CREB ShortCut small interfering RNA (siRNA), scramble sequence of siRNA, and transfection reagents were from New England Biolabs (Ipswich, MA); transfection reagents were obtained from Targeting System (Santee, CA); nitrite/nitrate fluorometric assay reagents were purchased from Cayman Chemical (Ann Arbor, MI); antibodies for eNOS, phospho-CREB, CREB, and β-actin were from Cell Signaling Technology (Beverly, MA); nitrocellulose membranes and protein assay kits were from Bio-Rad (Hercules, CA); genistein, protease, and phosphatase inhibitor cocktails, H89, P3115, PD98059, LY294002, and other general chemicals were from Sigma (St. Louis, MO).

Techniques: Expressing, Western Blot, Control

Journal: Endocrinology

Article Title: Phytoestrogen Genistein Up-Regulates Endothelial Nitric Oxide Synthase Expression Via Activation of cAMP Response Element-Binding Protein in Human Aortic Endothelial Cells

doi: 10.1210/en.2012-1076

Figure Lengend Snippet: Genistein (G)-enhanced eNOS protein expression and NO production are mediated via activation of the PKA/CREB cascade in HAEC. A, Different concentrations of AdPKI or heat-inactivated AdPKI virus (H) were transfected into HAEC for 24 h. Cell lysates were used to measure PKA activity using a nonradioactive PKA assay kit with negative (−) and positive (+) controls included. B and C, HAEC transfected with (+) or without (−) AdPKI were treated in the presence or absence of G (10 μ m ) for 5 d. Protein expression of eNOS in cell extracts was analyzed with Western blotting and normalized to β-actin content. Values (mean ± se ) of three separate experiments are expressed as fold of control, and a set of representative images and bar graph (mean ± se ) are shown (B). Nitrite/nitrate (NO x ) production stimulated with ionophore 23187 was measured using a fluorometric assay kit and normalized to protein content. Values (mean ± se ) of three separate experiments were expressed as fold of control (C). D, Confluent HAEC were serum starved in HBSS buffer for 30 min and followed by stimulation of various concentrations of G (0, 1, and 10 μ m ) for 15 min. PKA activity was measured by an assay kit. Values (mean ± se ) of three separate experiments are expressed as fold of control. *, P < 0.05 vs. vehicle alone-treated cells.

Article Snippet: Primary human aortic EC (HAEC) and endothelial growth supplements (EGM2) were purchased from Lonza Walkersville (Walkersville, MD); M199 media and fetal bovine serum (FBS) were obtained from Invitrogen (Carlsbad, CA); pGL2-eNOS promoter-luciferase plasmid (eNOS-Luc) was from Addgene, Inc. (Cambridge, MA); PKA activity and dual luciferase reporter assay kits were from Promega (Madison, WI); CREB ShortCut small interfering RNA (siRNA), scramble sequence of siRNA, and transfection reagents were from New England Biolabs (Ipswich, MA); transfection reagents were obtained from Targeting System (Santee, CA); nitrite/nitrate fluorometric assay reagents were purchased from Cayman Chemical (Ann Arbor, MI); antibodies for eNOS, phospho-CREB, CREB, and β-actin were from Cell Signaling Technology (Beverly, MA); nitrocellulose membranes and protein assay kits were from Bio-Rad (Hercules, CA); genistein, protease, and phosphatase inhibitor cocktails, H89, P3115, PD98059, LY294002, and other general chemicals were from Sigma (St. Louis, MO).

Techniques: Expressing, Activation Assay, Virus, Transfection, Activity Assay, Protein Kinase A Assay, Western Blot, Control

Journal: Endocrinology

Article Title: Phytoestrogen Genistein Up-Regulates Endothelial Nitric Oxide Synthase Expression Via Activation of cAMP Response Element-Binding Protein in Human Aortic Endothelial Cells

doi: 10.1210/en.2012-1076

Figure Lengend Snippet: Genistein (G)-stimulated eNOS expression and NO production are mediated via CREB. A and B, Confluent HAEC were incubated in HBSS buffer (A) or preincubated with H89 in HBSS buffer (B) for 30 min followed by the addition of various concentrations of G (0.01–10 μ m ) for 15 min. CREB phosphorylation was measured by Western blotting. C–E, CREB siRNA or scramble sequence of siRNA was transfected into HAEC for 24 h. Cells were then treated with G (0, 1, and 10 μ m ) for 15 min in HBSS buffer for determining CREB phosphorylation and expression (C) or 5 d in EGM2 medium for determining eNOS protein expression (D) and NO production (E) as described in Materials and Methods . Values (mean ± se ) of three separate experiments are expressed as fold over control; *, P < 0.05 vs. vehicle alone-treated control.

Article Snippet: Primary human aortic EC (HAEC) and endothelial growth supplements (EGM2) were purchased from Lonza Walkersville (Walkersville, MD); M199 media and fetal bovine serum (FBS) were obtained from Invitrogen (Carlsbad, CA); pGL2-eNOS promoter-luciferase plasmid (eNOS-Luc) was from Addgene, Inc. (Cambridge, MA); PKA activity and dual luciferase reporter assay kits were from Promega (Madison, WI); CREB ShortCut small interfering RNA (siRNA), scramble sequence of siRNA, and transfection reagents were from New England Biolabs (Ipswich, MA); transfection reagents were obtained from Targeting System (Santee, CA); nitrite/nitrate fluorometric assay reagents were purchased from Cayman Chemical (Ann Arbor, MI); antibodies for eNOS, phospho-CREB, CREB, and β-actin were from Cell Signaling Technology (Beverly, MA); nitrocellulose membranes and protein assay kits were from Bio-Rad (Hercules, CA); genistein, protease, and phosphatase inhibitor cocktails, H89, P3115, PD98059, LY294002, and other general chemicals were from Sigma (St. Louis, MO).

Techniques: Expressing, Incubation, Phospho-proteomics, Western Blot, Sequencing, Transfection, Control

Journal: Biochemical Journal

Article Title: Arginine promotes the activation of human lung fibroblasts independent of its metabolism

doi: 10.1042/BCJ20253033

Figure Lengend Snippet: ( A ) Schematic illustration of arginine metabolism. Arginine is catabolized by arginase 1 and 2 (ARG1, ARG2) or by nitric oxide synthase (NOS), producing urea or nitric oxide, respectively. In cells with a functional urea cycle, ornithine is converted to citrulline by ornithine transcarbamylase. Citrulline is converted back to arginine through the combined actions of argininosuccinate synthase 1 (ASS1) and argininosuccinate lyase (ASL). ( B ) Intracellular levels of arginine, ornithine and citrulline in HLFs cultured in DMEM or Human Plasma-Like Medium (HPLM) and treated with TGF-β or left untreated for 48 hours. ( C ) Intracellular levels of arginine, ornithine, and citrulline from three clones of normal and three clones of IPF HLFs. Cells were cultured in HPLM and treated with TGF-β or left untreated for 48 hours. ( D ) Heatmap analysis showing the Kyoto Encyclopedia of Genes and Genomes (KEGG) arginine and proline metabolism pathway on RNA-seq data from three clones of normal and three clones of IPF HLFs (three biological replicates per clone). Cells were cultured in HPLM and treated with TGF-β or left untreated for 24 hours. ( E ) Western blot analysis showing protein expression of ASS1, ASL, ARG2, and NOS3 in three clones of normal and three clones of IPF HLFs. Cells were cultured in HPLM and treated with TGF-β for the indicated intervals.

Article Snippet: Primary antibodies used were as follows: Collagen 1 (Abcam, ab138492), α-SMA (Sigma, A2547), Phospho-GCN2 (Cell Signaling, 94668), GCN2 (Cell Signaling, 3302), Phospho-P70S6K (Cell Signaling, 9234), P70S6K (Cell Signaling, 9202), Phospho-SMAD2/3 (Cell Signaling, 9520), SMAD2/3 (Cell Signaling, 9523), ASS1 (Invitrogen, PA-5-82740), ASL (Novus, NBP1-87462), NOS3 (Proteintech, 27120-1-AP), ARG2 (Invitrogen PA5-78820), TβR1 (Cell Signaling, 49728), TβR2 (Cell Signaling, 41896), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling, 2118).

Techniques: Functional Assay, Cell Culture, Clinical Proteomics, Clone Assay, RNA Sequencing, Western Blot, Expressing

Journal: The Journal of investigative dermatology

Article Title: Loss of Endothelial Cell Matrix Metalloproteinase 14 Reduces Melanoma Growth and Metastasis by Increasing Tumor Vessel Stability.

doi: 10.1016/j.jid.2021.12.016

Figure Lengend Snippet: Figure 6. eNOS expression and NO production in EC. (a) Analysis of DAF-FM fluorescence intensity of primary ECs measured at 495 nm. Scatter dot plot shows the mean SD; n ¼ 3. **P < 0.005. (b) NOS3 transcript amplification in primary ECs of Mmp14fl/fland Mmp14EC‒/‒ mice using S26 as control. Scatter dot plot shows the mean SD; n ¼ 9; *P < 0.05. (c) Primary ECs stained for eNOS (red) and nuclei (DAPI, blue). eNOS expression intensity was quantified. The scatter dot plot shows the mean SD. **P < 0.005. Bar ¼ 50 mm. (d) Quantitative analysis of eNOS and MMP14 expression in human melanoma. Intensities are arbitrarily set as þ (low), þþ (moderate), and þþþ (high). Graphs show the percentage of eNOS expression intensity in low, moderate, or high MMP14- expressing vessels. Arrowheads indicate representative vessels. n ¼ 21. Bar ¼ 250 mm. DAF-FM, diaminofluorescein-FM; EC, endothelial cell; eNOS, endothelial nitric oxide synthase; MMP14, matrix metalloproteinase 14; NO, nitric oxide.

Article Snippet: The small interfering RNA used for mouse eNOS (NOS3) was from Santa Cruz Biotechnology (Heidelberg, Germany) (sc36094).

Techniques: Expressing, Control, Staining