Journal: Annals of Medicine

Article Title: LPI-GPR55 promotes endothelial cell activation and inhibits autophagy through inducing LINC01235 expression

doi: 10.1080/07853890.2024.2407525

Figure Lengend Snippet: LPI targeted GPR55 and promoted endothelial cell activation. (a, b) Western blot analysis of GPR55 and ICAM1 protein levels after treating with LPI (1, 5 and 10 μM) for 18 h. (c, d) RT-PCR analysis of ICAM1 and GPR55 mRNA levels after treating with LPI (1, 5 and 10 μM) for 18 h. (e, g) HAECs were transfected with siGPR55 at 20 and 60 nM for 24 h. RT-PCR and Western blot analysed the RNA and protein level of GPR55. (f) HAECs were transfected with siGPR55 (60 nM) for 24 h and then treated with LPI (10 μM) for 18 h. Western blot analysed ICAM1 protein level. (h) Immunofluorescence analysed the adhesion of monocytes to endothelial cells after treating with LPI (10 μM) for 18 h, scar bar: 500 μm. Data are represented as mean ± SD (* p < .05, ** p < .01 and *** p < .001, n = 3).

Article Snippet: In this study, we used human aortic endothelial cells (HAECs: ATCC ® PCS-100-011, accession no. CVCL_C0EQ) as VEC model. HAECs were grown in vascular cell basal medium (ATCC ® PCS-100-030) with an endothelial cell growth kit-VEGF (ATCC ® PCS-100-041) in a humidified incubator at 37 °C with 5% CO 2 .

Techniques: Activation Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, Immunofluorescence

Journal: Cell Reports

Article Title: Luminescent sensing of conformational integrin activation in living cells

doi: 10.1016/j.celrep.2025.115319

Figure Lengend Snippet: β1IAS functional characterization in genetic β1IAS KI TeloHAECs (A) Western blot showing the expression of β1 integrin and NanoBiT tag in β1IAS knockin (KI) ECs compared to WT TeloHAECs (parental). (B) Relative adhesion measured by the xCELLigence system in β1IAS KI ECs plated on FN compared to parental ECs. Data are the mean ± SD of four independent experiments. Statistical analysis: two-way ANOVA and Bonferroni’s post hoc analysis. (C) Luminescence of β1IAS KI ECs adhering for 30 min to increasing amounts (125–1,000 ng/mL) of FN, collagen type I (Coll I), or laminin 511 (Lam 511). Data are mean ± SD of three independent experiments. Statistical analysis: two-way ANOVA and Bonferroni’s post hoc analysis. (D) Luminescence of β1IAS KI ECs adhering for 30 min to increasing amounts (125–1,000 ng/mL) of vitronectin (VN). Data are mean ± SD of three independent experiments. Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis. (E) Luminescence of β1IAS KI ECs adhering for 30 min to FN-coated, increasingly stiff (10 and 100 kPa) PAGEs. Data are mean ± SD of four independent experiments. Statistical analysis: two-tailed heteroscedastic Student’s t-test. (F) Luminescence of β1IAS KI ECs adhering for 15 min to 500 ng/mL FN in the presence of 9EG7 and 12G10 antibodies. Data are mean ± SD of three independent experiments. Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis. (G) Luminescence of β1IAS KI ECs adhering for 15 min to 500 ng/mL FN in the presence of mAb13 or the pan-αv integrin and α5β1 antagonist MK-0429 (100 μM). Data are mean ± SD of three independent experiments. Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis. (H) Luminescence of control β1IAS KI ECs (siCTL) and β1IAS KI ECs silenced for TLN1, FERMT2, and FERMT3 and then plated on 500 ng/mL FN. Data are mean ± SD of three independent experiments. Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis. (I) Luminescence of control β1IAS KI ECs (siCTL) and β1IAS KI ECs silenced for FLRT2, LPHN2, and PLXND1 and then plated on 500 ng/mL FN. Data are mean ± SD of three independent experiments. Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis.

Article Snippet: hTERT-immortalized human aortic endothelial cells (TeloHAECs) , ATCC , CRL-4052.

Techniques: Functional Assay, Western Blot, Expressing, Knock-In, Two Tailed Test, Control

Journal: Cell Reports

Article Title: Luminescent sensing of conformational integrin activation in living cells

doi: 10.1016/j.celrep.2025.115319

Figure Lengend Snippet: siRNA HTS in β1IAS TeloHAECs to identify activators and inhibitors of β1 integrin (A) Schematic of the high-throughput screening (HTS)-selected genes (194), among the 2,970 genes expressed by parental WT TeloHAECs, whose silencing in β1IAS KI ECs induces a decreased luminescent signal ( Z score < −2), therefore β1 integrin activators (142, in green), and those whose silencing induces an increased luminescent signal ( Z score > 2), therefore β1 integrin inhibitors (52, in red). (B) Bubble plot representing top integrin focused enriched pathways (adjusted p < 0.05) based on candidate genes obtained from the HTS (2 < Z score < −2). All enriched pathways are listed in Table S2 . The EnrichR combined score is the log of the p value from the Fisher exact test multiplied by the Z score of the deviation from the expected rank. Bubble color (adjusted p value) was computed using the Benjamini-Hochberg method for correction for multiple hypotheses testing. The gene ratio is the overlap between the input list and the gene sets in each gene set library for ranking a pathway’s relevance to the input list. (C) Luminescent intensity Z score mean of three biological replicates for each endothelial gene whose siRNA was contained in the Qiagen Druggable Genome v.3 siRNA library. The listed genes (β1 integrin inhibitors in red and β1 integrin activators in green) were chosen for secondary validation. (D) Luminescence of control β1IAS KI ECs (siCTL) and β1IAS KI ECs silenced for RAP1B, TNS3, and RHOJ and then plated on 500 ng/mL FN. Data are mean ± SD of three independent experiments. Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis. (E) Luminescence of control β1IAS KI ECs (siCTL) and β1IAS KI ECs silenced for RACGAP1, PJA2, and VEGF-B and then plated on 500 ng/mL FN. Data are mean ± SD of three independent experiments. Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis.

Article Snippet: hTERT-immortalized human aortic endothelial cells (TeloHAECs) , ATCC , CRL-4052.

Techniques: High Throughput Screening Assay, Biomarker Discovery, Control

Journal: Cell Reports

Article Title: Luminescent sensing of conformational integrin activation in living cells

doi: 10.1016/j.celrep.2025.115319

Figure Lengend Snippet: The E3 ubiquitin ligase PJA2 promotes kindlin-2 degradation and inhibits β1 integrin activation in ECs (A) Left: western blot showing the expression of PJA2 in WT TeloHAECs after PJA2 silencing and silenced cells transduced with silencing resistant murine PJA2 (mPJA2). Right: luminescence intensity of β1IAS KI ECs plated on 500 ng/mL FN, silenced for PJA2, and rescued with mPJA2. Data are mean ± SD of three independent experiments. Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis. (B) Western blot showing the expression of kindlin-2, talin-1, and Rap1B in WT TeloHAECs after PJA2 silencing. (C) Left: western blot showing ubiquitinated GFP kindlin-2 pulled down by ubiquitin affinity beads in Phoenix cells overexpressing PJA2 or control construct. The first lane corresponds to the incubation of lysate from cells overexpressing control constructs on non-ubiquitinated beads (see ). Right: western blot showing the expression of PJA2 in cells used in the ubiquitinated assay shown on the left. (D) Confocal microscopy showing 9EG7 + active β1 integrin (green), kindlin-2 (red) and vinculin (blue) in WT TeloHAECs plated on 1.5 μg/mL FN and silenced for PJA2. The image insets highlight focal adhesion sites. Scale bar: 20 μm. (E) Relative maximum Feret diameter (mFD) of adhesion sites (FA) in WT TeloHAECs silenced for PJA2 compared to siCTL. Data are the mean ± SD of three independent experiments (10 cells each). Statistical analysis: two-tailed heteroscedastic Student’s t test. (F) Number of adhesion sites (FA) in siCTL and siPJA2 ECs as in (E). Data are the mean ± SD of three independent experiments (9 cells each). Statistical analysis: two-tailed heteroscedastic Student’s t test.

Article Snippet: hTERT-immortalized human aortic endothelial cells (TeloHAECs) , ATCC , CRL-4052.

Techniques: Ubiquitin Proteomics, Activation Assay, Western Blot, Expressing, Transduction, Control, Construct, Incubation, Confocal Microscopy, Two Tailed Test

Journal: Cell Reports

Article Title: Luminescent sensing of conformational integrin activation in living cells

doi: 10.1016/j.celrep.2025.115319

Figure Lengend Snippet: VEGF-B is an effective inhibitor of β1 integrin activation in ECs (A) Left: western blot showing the expression of VEGF-B protein in WT TeloHAECs after VEGFB silencing compared to siCTL. Right: luminescence of TeloHAEC β1IAS plated on 500 ng/mL FN and silenced for VEGFB in the presence or absence of exogenous VEGF-B for 15 min. Data are mean ± SD of three independent experiments. Statistical analysis: one-way ANOVA and Bonferroni’s post hoc analysis. (B) Confocal microscopy showing 9EG7 + active β1 integrin (green), kindlin-2 (red), and vinculin (blue) in WT TeloHAECs plated on 1.5 μg/mL FN and silenced for VEGFB. The image insets highlight focal adhesion sites. Scale bar: 20 μm. (C) Relative maximum Feret diameter (mFD) of adhesion sites (FA) in WT TeloHAECs silenced for VEGFB compared to siCTL. Data are the mean ± SD of three independent experiments (10 cells each). Statistical analysis: two-tailed heteroscedastic Student’s t test. (D) Number of adhesion sites (FA) in WT TeloHAECs silenced for VEGFB compared to siCTL. Data are the mean ± SD of three independent experiments (9 cells each). Statistical analysis: two-tailed heteroscedastic Student’s t test. (E) Relative adhesion measured by the xCELLigence system in WT TeloHAECs plated on 1.5 μg/mL FN and silenced for VEGFB. Data are the mean ± SD of five independent experiments. Statistical analysis: two-way ANOVA and Bonferroni’s post hoc analysis. (F) Relative adhesion measured by the xCELLigence system in WT TeloHAECs plated on 1.5 μg/mL FN and treated or not with 100 and 200 ng/mL exogenous VEGF-B. Data are the mean ± SD of three independent experiments. Statistical analysis: two-way ANOVA and Bonferroni’s post hoc analysis.

Article Snippet: hTERT-immortalized human aortic endothelial cells (TeloHAECs) , ATCC , CRL-4052.

Techniques: Activation Assay, Western Blot, Expressing, Confocal Microscopy, Two Tailed Test

Journal: Cell Reports

Article Title: Luminescent sensing of conformational integrin activation in living cells

doi: 10.1016/j.celrep.2025.115319

Figure Lengend Snippet: VEGF-B modulates the phosphorylation of CMSC mediators (A and B) Volcano plots of the phosphoproteome of parental WT TeloHAECs stimulated with VEGF-B or control for 15 (A) or 30 (B) min. n = 4 biological replicates. Colored dots are phosphorylation sites of proteins annotated to the GOBP categories “cell adhesion” or “cytoskeleton organization.” Dashed bars separate significantly regulated sites with p ≤ 0.05 and difference ≥ ±0.2. The position of the phosphorylation site within the protein sequence is in parentheses following the gene name. (C and D) Heatmaps of the up- and downregulated sites highlighted in the volcano plots in (A) and (B), respectively. Colors are based on the intensity values measured for the phosphorylated peptide by MaxQuant; purple represents upregulation upon VEGF-B stimulation, and green represents downregulation upon VEGF-B stimulation. An ∗ indicates a known regulatory site. (E) Left: confocal microscopy showing talin-1 (green) and KANK3 (red) in WT TeloHAECs plated on 1.5 μg/mL FN and silenced for VEGFB. The image insets highlight contact sites between talin-1 + adhesions and the CMSC mediator KANK3. Scale bar: 20 μm. Right: Pearson correlation between talin-1 + adhesions and the CMSC mediator KANK3. Data are the mean ± SD of two independent experiments (10 cells each). Statistical analysis: two-tailed heteroscedastic Student’s t test.

Article Snippet: hTERT-immortalized human aortic endothelial cells (TeloHAECs) , ATCC , CRL-4052.

Techniques: Phospho-proteomics, Control, Sequencing, Confocal Microscopy, Two Tailed Test

Journal: Cell Reports

Article Title: Luminescent sensing of conformational integrin activation in living cells

doi: 10.1016/j.celrep.2025.115319

Figure Lengend Snippet:

Article Snippet: hTERT-immortalized human aortic endothelial cells (TeloHAECs) , ATCC , CRL-4052.

Techniques: Recombinant, Fluorescence, Ubiquitin Proteomics, Bicinchoninic Acid Protein Assay, Magnetic Beads, Plasmid Preparation, Software

Journal: Free radical biology & medicine

Article Title: Nitroglycerin drives endothelial nitric oxide synthase activation via the phosphatidylinositol 3-kinase/protein kinase B pathway

doi: 10.1016/j.freeradbiomed.2011.09.020

Figure Lengend Snippet: PI3K involvement in GTN-dependent vasodilation. (A) Effect of wortmannin (PI3K inhibitor) pretreatment upon the acetylcholine (Ach)- or GTN-induced dilation of rat aortic rings. (B) Nitroglycerin EC50 values measured in rat aortic rings in the presence of the indicated concentrations of Akt 1/2 inhibitor. (C) Vasodilation experiment with mesenteric tissue recovered from p110γ-knockout mice. p110γ is the catalytic subunit of endothelial PI3K enzyme. Both pharmacologic inhibition and genetic knockout of PI3K inhibited GTN-induced dilation of conducing (aorta) and resistant (mesenteric artery) vessels. *P<0.05, **P<0.01.

Article Snippet: Human and bovine aortic endothelial cells (HAEC or BAEC; Allcells, Emeryville, CA, USA), human microvascular endothelial cells (HMEC), and primary mouse endothelial cells (MEC) were cultured at 37 °C, 5% CO 2 in corresponding endothelial medium with supplements including growth factors, 10% FBS, and 1% antibiotics–antimycotics (Sigma).

Techniques: Knock-Out, Inhibition

Journal: Free radical biology & medicine

Article Title: Nitroglycerin drives endothelial nitric oxide synthase activation via the phosphatidylinositol 3-kinase/protein kinase B pathway

doi: 10.1016/j.freeradbiomed.2011.09.020

Figure Lengend Snippet: PI3K/Akt-dependent phosphorylation of eNOS elicited by nitroglycerin. BAEC were challenged with nitroglycerin (500 nM) for 3 min before harvest. PI3K inhibitor wortmannin (100 nM) or Akt inhibitor (20 µM) was dissolved in DMSO and added to the cell cultures 1 h before GTN treatment; DMSO was also added at the same concentration (v/v) to control groups. Final DMSO concentration did not exceed 0.1%.

Article Snippet: Human and bovine aortic endothelial cells (HAEC or BAEC; Allcells, Emeryville, CA, USA), human microvascular endothelial cells (HMEC), and primary mouse endothelial cells (MEC) were cultured at 37 °C, 5% CO 2 in corresponding endothelial medium with supplements including growth factors, 10% FBS, and 1% antibiotics–antimycotics (Sigma).

Techniques: Phospho-proteomics, Concentration Assay, Control

Journal: Free radical biology & medicine

Article Title: Nitroglycerin drives endothelial nitric oxide synthase activation via the phosphatidylinositol 3-kinase/protein kinase B pathway

doi: 10.1016/j.freeradbiomed.2011.09.020

Figure Lengend Snippet: Time-dependent activation of Akt and eNOS paralleling PTEN phosphorylation by GTN. Representative Western blots showing PTEN phosphorylation (Ser 380), eNOS phosphorylation (Ser 1177 or 1179), and Akt phosphorylation (Ser 473) in (A) BAEC and (B) HMEC treated with vehicle or 500 nM GTN for the indicated amounts of time. Vehicle was added for 10 min in (A) and 15 min in (B). Results show rapid and sustained eNOS phosphorylation at the activation site Ser 1177, Akt phosphorylation at the activation site Ser 473, and PTEN phosphorylation at the inhibitory site Ser 380 by 500 nM GTN. Band intensities for phosphorylated eNOS in BAEC and for the experiments performed with HMEC were quantified using ImageJ and the values are reported as density units relative to control; *P<0.05, **P<0.01.

Article Snippet: Human and bovine aortic endothelial cells (HAEC or BAEC; Allcells, Emeryville, CA, USA), human microvascular endothelial cells (HMEC), and primary mouse endothelial cells (MEC) were cultured at 37 °C, 5% CO 2 in corresponding endothelial medium with supplements including growth factors, 10% FBS, and 1% antibiotics–antimycotics (Sigma).

Techniques: Activation Assay, Phospho-proteomics, Western Blot, Control

Journal: Free radical biology & medicine

Article Title: Nitroglycerin drives endothelial nitric oxide synthase activation via the phosphatidylinositol 3-kinase/protein kinase B pathway

doi: 10.1016/j.freeradbiomed.2011.09.020

Figure Lengend Snippet: Concentration-dependent Akt activation by GTN. (A) BAEC cells were treated with vehicle control (30% ethanol, 30% propylene glycol, 40% water) or the indicated GTN concentrations for 3 min before harvest. (B) Band densities were measured using ImageJ software. Results were quantified as relative Akt activation compared with control; *P<0.05 by Student's t test.

Article Snippet: Human and bovine aortic endothelial cells (HAEC or BAEC; Allcells, Emeryville, CA, USA), human microvascular endothelial cells (HMEC), and primary mouse endothelial cells (MEC) were cultured at 37 °C, 5% CO 2 in corresponding endothelial medium with supplements including growth factors, 10% FBS, and 1% antibiotics–antimycotics (Sigma).

Techniques: Concentration Assay, Activation Assay, Control, Software