Journal: Molecular Biology of the Cell

Article Title: Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition

doi: 10.1091/mbc.e11-09-0811

Figure Lengend Snippet: FIGURE 1: Interaction of Cav-1 and eNOS determined by FRET and coIP. (A) Typical FRET image of Cav-1–YFP and eNOS–CFP expressed in CHO cells. Note that the fluorescence intensity of YFP increases, whereas CFP decreases in the ROI (white box) after addition of 5 μM Ca2+ ionophore A23187. Bar, 10 μm. (B) Normalized FRET measurements in CHO cells expressing Cav-1–YFP and eNOS–CFP after treatment with 5 μM A23187 (mean ± SEM; n = 12) or 4 U/ml thrombin (mean ± SEM; n = 5). YFP/CFP was normalized as 1 by the value at time zero (*p < 0.01 for all times beyond 2 min after thrombin addition; **p < 0.005 for all times beyond 1.5 min after A23187). (C) CoIP between Cav-1–YFP and eNOS–CFP in CHO cells (mean ± SEM; n = 3). (D) Normalized data of coIP (ratio of eNOS-CFP/Cav-1-YFP after IP with anti–Cav-1 antibody; *p < 0.05). (E) Phosphorylation of Cav-1–YFP and eNOS–CFP in CHO cells after treatment with 5 μM A23187 or 4 U/ml thrombin (mean ± SEM; n = 3). Ratio of phosphorylated/ total protein at 0 min was set as 1 (*p < 0.05).

Article Snippet: To create the vector eNOS with CFP tagged at the C-terminus, full-length H. sapiens eNOS cDNA (with GenBank Accession number NM_000603 from OriGene Technologies, Rockville, MD) was used as DNA template in a PCR to generate eNOS PCR fragment that contained AgeI at both 5′ and 3′ ends.

Techniques: Fluorescence, Expressing, Phospho-proteomics

Journal: Molecular Biology of the Cell

Article Title: Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition

doi: 10.1091/mbc.e11-09-0811

Figure Lengend Snippet: FIGURE 2: CoIP between eNOS and Cav-1 in mouse lung. (A) Mouse lungs were perfused with medium containing 5 μM A23187 for 0, 3, and 10 min. Lungs were then homogenized and prepared for IP with polyclonal anti–Cav-1. (B) Normalized coIP data (mean ± SEM; n = 4); ratio of eNOS/Cav-1 at 0 min was set as 1 (*p < 0.05). (C) Phosphorylation of eNOS–Ser-1177, Src–Tyr-418, and Cav-1–Tyr-14 (indicated by arrows) in mouse lung homogenates following A23187 treatment (D). Normalized ratio of phosphorylated/total protein, with time 0 set as 1 (*p < 0.05; mean ± SEM; n = 4).

Article Snippet: To create the vector eNOS with CFP tagged at the C-terminus, full-length H. sapiens eNOS cDNA (with GenBank Accession number NM_000603 from OriGene Technologies, Rockville, MD) was used as DNA template in a PCR to generate eNOS PCR fragment that contained AgeI at both 5′ and 3′ ends.

Techniques: Phospho-proteomics

Journal: Molecular Biology of the Cell

Article Title: Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition

doi: 10.1091/mbc.e11-09-0811

Figure Lengend Snippet: FIGURE 3: Phosphorylation-dependent interaction of eNOS and Cav-1 in HUVEC. (A) Time course of phosphorylation of eNOS–Ser-1177 and Cav-1–Tyr-14 in HUVECs after addition of 5 μM A23187, 4 U/ml thrombin, or 20 ng/ml VEGF (mean ± SEM; n = 6). Normalized data are shown in the bottom two rows (*p < 0.05). (B) CoIP of eNOS and Cav-1 in HUVEC after stimulation with 5 μM A23187 at indicated times (mean ± SEM; n = 4. *p < 0.05; **p < 0.005). (C, D) CoIP of phosphorylated eNOS and Cav-1 in HUVECs treated with A23187 for 5 min at 37°C (mean ± SEM; n = 3). p-Ser-1177-eNOS antibody was used for IP. Normalized data (p-Tyr-14-Cav-1 vs. t-Cav-1 bound to p-eNOS) is shown in D (*p < 0.05). (E) Effects of l-NAME and PP2 on phosphorylation of eNOS–Ser-1177, Src–Tyr-418, and Cav-1–Tyr-14 in HUVECs (mean ± SEM; n = 9). Confluent HUVECs were pretreated with 1 mM l-NAME or 15 μM PP2 for 30 min at 37°C prior to addition of 5 μM A23187 (§p < 0.001; *p < 0.05; and **p < 0.001). (F) Effect of l-NAME and PP2 on coIP between eNOS and Cav-1 (mean ± SEM; n = 6) in HUVEC (*p < 0.05; **p < 0.005).

Article Snippet: To create the vector eNOS with CFP tagged at the C-terminus, full-length H. sapiens eNOS cDNA (with GenBank Accession number NM_000603 from OriGene Technologies, Rockville, MD) was used as DNA template in a PCR to generate eNOS PCR fragment that contained AgeI at both 5′ and 3′ ends.

Techniques: Phospho-proteomics

Journal: Molecular Biology of the Cell

Article Title: Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition

doi: 10.1091/mbc.e11-09-0811

Figure Lengend Snippet: FIGURE 4: Binding of Y14D-Cav-1 with eNOS inhibits eNOS activity. (A) CoIP of Cav-1 and eNOS mutants in HEK cells. WT-eNOS-CFP, eNOS-S1177D-CFP, and eNOS-S1177A-CFP were transiently transfected in HEK cells stably expressing WT-Cav-1-YFP, Cav-1-Y14D-YFP, or Cav-1-Y14F-YFP. Cell lysates were immunoprecipitated with anti–Cav-1 pAb 48 h after transfection of eNOS mutants in the absence of stimulation. Ratio of WT-eNOS-CFP/wt-Cav-1- YFP was set at 1 (*p < 0.05; **p < 0.005). Bar graph shows mean ± SEM (n = 3). (B) Protein levels of Cav-1 and eNOS mutants in transfected HEK cells before IP. (C) Nitrite accumulation in HEK/ eNOS cells transiently transfected with Cav-1 mutant cDNAs (mean ± SEM; n = 11). Cells were treated for 30 min with 5 μM A23187, and supernatants were collected for nitrite assay (*p < 0.001). (D) eNOS activity (representative of four independent experiments) of stable HEK/ eNOS cells transiently transfected with Cav-1 mutants.

Article Snippet: To create the vector eNOS with CFP tagged at the C-terminus, full-length H. sapiens eNOS cDNA (with GenBank Accession number NM_000603 from OriGene Technologies, Rockville, MD) was used as DNA template in a PCR to generate eNOS PCR fragment that contained AgeI at both 5′ and 3′ ends.

Techniques: Binding Assay, Activity Assay, Transfection, Stable Transfection, Expressing, Immunoprecipitation, Mutagenesis, Nitration

Journal: Molecular Biology of the Cell

Article Title: Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition

doi: 10.1091/mbc.e11-09-0811

Figure Lengend Snippet: FIGURE 5: NO-dependent Src activation mediates Cav-1–Tyr-14 phosphorylation. (A) Decreased phosphorylation of Src–Tyr-418 and Cav-1–Tyr-14 in HUVECs after eNOS siRNA treatment (mean ± SEM; n = 4). Ratio of phosphorylated to total protein at 0 min was set at 1 (*p < 0.05). (B) The NO donor DEA NONOate induces Src activation in Cav-1–null MEF cells (mean ± SEM; n = 3). After stimulation with 0.2 mM DEA NONOate at indicated times, cells were prepared for Western blotting (*p < 0.05). (C) eNOS/NO-dependent Src activation in HEK cells (mean ± SEM; n = 7). The ratio of p-Tyr-418-Src at 5 min of A23187 treatment vs. untreated in WT-HEK cells was set as 1 (*p < 0.001 vs. WT-HEK). (D) A typical live-cell FRET image of HEK/eNOS cells expressing Src FRET biosensor. Fluorescence intensity of CFP and YFP was measured in the ROI (white box) before and after treatment with 5 μM A23187. Bar, 10 μm. (E) Activation of Src measured by FRET in HEK/eNOS cells expressing Src-FRET biosensor (mean ± SEM; n = 13). After cells were pretreated with medium alone, 1 mM l-NAME, or 15 μM PP2 for 30 min at 37°C, 5 μM A23187 was added and FRET was measured (*p < 0.05 vs. WT-HEK cells; **p < 0.01 all times beyond 2 min after A23187 addition vs. 0 min).

Article Snippet: To create the vector eNOS with CFP tagged at the C-terminus, full-length H. sapiens eNOS cDNA (with GenBank Accession number NM_000603 from OriGene Technologies, Rockville, MD) was used as DNA template in a PCR to generate eNOS PCR fragment that contained AgeI at both 5′ and 3′ ends.

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Expressing, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: Nitric oxide–dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition

doi: 10.1091/mbc.e11-09-0811

Figure Lengend Snippet: FIGURE 6: Proposed mechanism of negative feedback regulation of eNOS by phospho–Cav-1. Ca2+-dependent stimulation of endothelial cells induces eNOS activation by phosphorylation on Ser-1177, and NO is released within 1 min. NO induces Src activation and phosphorylation of Cav-1 on Tyr-14, which we propose leads to unmasking of the caveolin scaffolding domain. Phosphorylated Cav-1 then binds to activated eNOS, inhibiting eNOS activity, and release of NO, thus returning eNOS to its basal state. eNOS/Cav-1 interaction can be blocked by eNOS inhibitor l-NAME and Src inhibitor PP2, suggesting that an NO/Src-dependent feedback mechanism mediates eNOS inactivation.

Article Snippet: To create the vector eNOS with CFP tagged at the C-terminus, full-length H. sapiens eNOS cDNA (with GenBank Accession number NM_000603 from OriGene Technologies, Rockville, MD) was used as DNA template in a PCR to generate eNOS PCR fragment that contained AgeI at both 5′ and 3′ ends.

Techniques: Activation Assay, Phospho-proteomics, Scaffolding, Activity Assay

Journal: Human Cell

Article Title: Single-cell dynamic RNA and glycosylation sequencing reveals the mechanism underlying the differentiation of pluripotent stem cells into hematopoietic stem cells

doi: 10.1007/s13577-025-01234-7

Figure Lengend Snippet: The analysis for the heterogeneity of endothelial clusters. a . The gene expression of endothelial, angiogenesis, and hematopoiesis in new RNA. b . The hotspot analyzed the similarity of cells and obtained 12 modules. c–d . The vascular endothelial cell migration pathway was selected from modules 3 and 8 and mapped to UMAP plots. e . The GO analysis of differential genes in high-glucose and low-glucose cells. f . The endothelial and hematopoietic genes in high-glucose and low-glucose cells in clusters 1 and 15

Article Snippet: From day 2 to day 6, 50 ng/mL human vascular endothelial growth factor (VEGF, MCE Catalog #HY- P70458 ), 50 ng/mL human basic fibroblast growth factor (bFGF, MCE Catalog #HY-P7331), 5 ng/mL BMP4, and 10 μM SB-431542 (MCE Catalog #HY-10431) were added to the BDM.

Techniques: Gene Expression, Migration

Journal: Human Cell

Article Title: Single-cell dynamic RNA and glycosylation sequencing reveals the mechanism underlying the differentiation of pluripotent stem cells into hematopoietic stem cells

doi: 10.1007/s13577-025-01234-7

Figure Lengend Snippet: Potential linkages of endothelial subsets in their differentiation fate. a . Clusters 1 and 15 were reclustered to seven new clusters, which were identified by differentially expressed genes, including NEFH, CLDN5, TOP2 A, RUNX1, MCM3, ANGPT2, and IGFBP3. b . The expression of endothelial and hematopoietic-related genes in endothelial subsets. c–d . The NTR and the levels of glycation in endothelial subsets. e . The expression of transcription factors in the endothelial subsets. f . The correlation between clusters 1, 15, and the endothelial subsets using the Spearman correlation analysis

Article Snippet: From day 2 to day 6, 50 ng/mL human vascular endothelial growth factor (VEGF, MCE Catalog #HY- P70458 ), 50 ng/mL human basic fibroblast growth factor (bFGF, MCE Catalog #HY-P7331), 5 ng/mL BMP4, and 10 μM SB-431542 (MCE Catalog #HY-10431) were added to the BDM.

Techniques: Expressing

Journal: International journal of molecular medicine

Article Title: Naringenin modulates the NO‑cGMP‑PKG signaling pathway by binding to AKT to enhance osteogenic differentiation in hPDLSCs.

doi: 10.3892/ijmm.2024.5391

Figure Lengend Snippet: Figure 3. Ability of NAR to promote osteogenesis in human periodontal ligament stem cells is mitigated after L‑NAME (eNOS inhibitor) treatment. (A) Alkaline phosphatase staining in different treatment groups. (B) Alizarin red staining in different treatment groups. (C) NO levels in different treatment groups. (D) Gene expression levels of RUNX2, OPN and sGC in different treatment groups. (E‑G) Protein expression of RUNX2, OPN, sGC and p‑eNOS in various treatment groups. *P<0.05, **P<0.01, ***P<0.001. NAR, naringenin; eNOS, endothelial nitric oxide synthase; RUNX2, Runt‑related transcription factor; OPN, osteopontin; sGC, soluble guanylate cyclase; NO, nitric oxide; p‑, phosphorylated; t‑, total; L‑NAME, NG‑nitro‑L‑arginine methyl ester.

Article Snippet: Cells treated with NAR (10 μmol/l) were also treated with NG‐nitro‐L‐arginine methyl ester (L‐NAME; 70 μM) to inhibit endothelial nitric oxide synthase (eNOS), 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ; 20 μM) to inhibit soluble guanylate cyclase (sGC) and AKT inhibitor VIII (58 nM) to inhibit AKT, as recommended by the manufacturer (MedChemExpress for all).

Techniques: Staining, Gene Expression, Expressing

Journal: International journal of molecular medicine

Article Title: Naringenin modulates the NO‑cGMP‑PKG signaling pathway by binding to AKT to enhance osteogenic differentiation in hPDLSCs.

doi: 10.3892/ijmm.2024.5391

Figure Lengend Snippet: Figure 6. Impact of AKT inhibitor VIII on NAR‑mediated osteogenic promotion in human periodontal ligament stem cells. (A) Alkaline phosphatase staining in different treatment groups. (B) Alizarin red staining in different treatment groups. (C) Gene expression of RUNX2 and OPN in different treatment groups. (D‑F) Protein levels of RUNX2, OPN, p‑AKT and p‑eNOS in different treatment groups. *P<0.05, **P<0.01, ***P<0.001. NAR, naringenin; RUNX2, Runt‑related transcription factor; OPN, osteopontin; eNOS, endothelial nitric oxide synthase; p‑, phosphorylated; t‑, total.

Article Snippet: Cells treated with NAR (10 μmol/l) were also treated with NG‐nitro‐L‐arginine methyl ester (L‐NAME; 70 μM) to inhibit endothelial nitric oxide synthase (eNOS), 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ; 20 μM) to inhibit soluble guanylate cyclase (sGC) and AKT inhibitor VIII (58 nM) to inhibit AKT, as recommended by the manufacturer (MedChemExpress for all).

Techniques: Staining, Gene Expression

Journal: International journal of molecular medicine

Article Title: Naringenin modulates the NO‑cGMP‑PKG signaling pathway by binding to AKT to enhance osteogenic differentiation in hPDLSCs.

doi: 10.3892/ijmm.2024.5391

Figure Lengend Snippet: Figure 8. Schematic diagram of the mechanism by which naringenin promotes the osteogenic differentiation of human periodontal ligament stem cells. eNOS, endothelial nitric oxide synthase; p, phosphorylated; NO, nitric oxide; sGC, soluble guanylate cyclase; cGMP, cyclic guanosine monophosphate; TRPC6, transient receptor potential cation channel, subfamily C, member 6; PKG, protein kinase G.

Article Snippet: Cells treated with NAR (10 μmol/l) were also treated with NG‐nitro‐L‐arginine methyl ester (L‐NAME; 70 μM) to inhibit endothelial nitric oxide synthase (eNOS), 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ; 20 μM) to inhibit soluble guanylate cyclase (sGC) and AKT inhibitor VIII (58 nM) to inhibit AKT, as recommended by the manufacturer (MedChemExpress for all).

Techniques:

Journal: Current Research in Physiology

Article Title: The comparison of endothelial function of moderate intensity interval exercise with continuous exercise in healthy men

doi: 10.1016/j.crphys.2022.07.003

Figure Lengend Snippet: Comparison of vascular markers between intermittent and continuous exercise.

Article Snippet: Then, the enzyme-linked immunosorbent assay (ELISA) method was used for determination of serum concentrations of N-terminal proANP (NTproANP), N-terminal proBNP (NTproBNP), N-terminal proCNP (NTproCNP), endothelial nitric oxide synthase activity, endothelin 1 (ET-1), adiponectin, and leptin (Elabscience Cat. No: E-EL-H1848, Cat. No: E-EL-H0902, Cat. No: E-EL-H2538, Cat. No: E-EL-H0755, Cat. No: E-EL-H0064, Cat. No: E-EL-H0004, Cat. No: E-EL-H0113 respectively).

Techniques: Comparison, Activity Assay