Journal: Journal of Atherosclerosis and Thrombosis

Article Title: Vehicle-dependent Effects of Sphingosine 1-phosphate on Plasminogen Activator Inhibitor-1 Expression

doi: 10.5551/jat.37663

Figure Lengend Snippet: S1P bound to albumin induced PAI-1 expression by activating S1PR2 pathway in 3T3L1 adipocytes. (A) 3T3L1 adipocytes were pre-incubated for 18 hours in FBS-free DMEM and the medium was exchanged for FBS-free DMEM containing 20 µM of VPC23019, JTE013, or DMSO for 30 minutes. Then, the cells were challenged with a medium containing 10 µM of sphingosine 1-phosphate (S1P) with the corresponding inhibitors for 4 hours. The mRNA level of plasminogen activator inhibitor 1 (PAI-1) was analyzed using real-time PCR. 18S was utilized as an internal control ( n = 4–6/group). † p < 0.01 vs. DMSO alone, YC1 alone, S1P + VPC, and JTE alone, and p < 0.05 vs. S1P + JTE. ‡ p < 0.01 vs. other groups. (B, C). 3T3L1 adipocytes were pre-incubated for 18 hours in FBS-free DMEM, and the medium was exchanged for FBS-free DMEM with 10 µM of S1P bound to apoM-rich vehicle (ApoM) or control vehicle (Null) for 4 hours. Then, the modulation of the phosphorylation of moesin in 3T3L1 cells was examined using western blotting ( n = 6/group). ** p < 0.01, *** p < 0.001.

Article Snippet: VPC23019 (857360P; Avanti Polar Lipids, Alabaster, AL), JTE013 (10009458; Cayman Chemical, Ann Arbor, MI), Y27632 (257-00511; WAKO Pure Chemical Industries, Osaka, Japan), wortmannin, YC-1 (W1628, Y102; Sigma-Aldrich Co), and SIS3 and BAY11-7082 (sc-222318, sc-200615; Santa Cruz Biotechnology, Inc. TX) were dissolved in DMSO.

Techniques: Expressing, Incubation, Real-time Polymerase Chain Reaction, Control, Phospho-proteomics, Western Blot

Journal: Scientific Reports

Article Title: Thymosin β4 stabilizes hypoxia induced brain microvascular endothelial cell dysfunction through S1PR1 dependent mechanisms

doi: 10.1038/s41598-025-28435-2

Figure Lengend Snippet: Effect of VPC23019 (a S1PR1 inhibitor) and Tβ4 on S1PR1 and SGPL1 gene expression in hBMVEC. hBMVECs were pretreated with VPC2309 (50 nmol) for 2 h followed by 2 h with Tβ4 (1 μg/mL) and then was exposed to 2 h and 24 h hypoxia. The mRNA expression of ( A ) S1PR1 and ( B ) SGPL1 were measured using reverse transcription-quantitative PCR. ( C ) Representative image of the Western blot analysis of S1PR1 protein level. ( D ) Quantification of S1PR1 Western blot analysis. Results are presented as the mean ± SEM ( n = 3). * P < 0.05 vs. normoxia cells. # P < 0.05 vs. hypoxia cells.

Article Snippet: For VPC2309 (Cat no. sc-362817, Santa Cruz, USA), an inhibitor of S1PR1 treatment, the cells were pretreated with VPC2309 at a concentration of 50 nmol for 2 h prior to switching the cells in hypoxic chamber.

Techniques: Gene Expression, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

Journal: Scientific Reports

Article Title: Thymosin β4 stabilizes hypoxia induced brain microvascular endothelial cell dysfunction through S1PR1 dependent mechanisms

doi: 10.1038/s41598-025-28435-2

Figure Lengend Snippet: Effect of VPC23019 and Tβ4 on TJ genes expression in hBMVEC. hBMVECs were pretreated with VPC2309 (50 nmol) for 2 h followed by 2 h with Tβ4 (1 μg/mL) and then was exposed to 2 h and 24 h hypoxia. The mRNA expression of ( A ) Occludin, ( B ) Cldn 5 and ( C ) Tjp1 were measured using reverse transcription-quantitative PCR. ( D ) Representative image of the Western blot analysis of Cldn 5 and Occludin protein level. ( E ) The quantification of Cldn 5 and ( F ) Occludin Westerns were shown. These experiments were replicated three times, and amplifications were performed and normalized to GAPDH. Results are presented as the mean ± SEM ( n = 3). * P < 0.05 vs. normoxia cells. # P < 0.05 vs. hypoxia cells.

Article Snippet: For VPC2309 (Cat no. sc-362817, Santa Cruz, USA), an inhibitor of S1PR1 treatment, the cells were pretreated with VPC2309 at a concentration of 50 nmol for 2 h prior to switching the cells in hypoxic chamber.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Western Blot

Journal: Scientific Reports

Article Title: Thymosin β4 stabilizes hypoxia induced brain microvascular endothelial cell dysfunction through S1PR1 dependent mechanisms

doi: 10.1038/s41598-025-28435-2

Figure Lengend Snippet: Effect of VPC23019 and Tβ4 on hypoxia-induced hBMVECs monolayer permeability. Permeability assays were performed as described in legend of Fig. A. Cultured hBMVECs were pre-treated with VPC2309 for 2 h followed by 2 h Tβ4 (1 μg/mL) and subjected to 24 h hypoxia. The permeability results are presented as the mean ± SEM ( n = 3). * P < 0.05 vs. normoxia cells. # P < 0.05 vs. hypoxia cells. hBMVECs, human brain microvascular endothelial cells.

Article Snippet: For VPC2309 (Cat no. sc-362817, Santa Cruz, USA), an inhibitor of S1PR1 treatment, the cells were pretreated with VPC2309 at a concentration of 50 nmol for 2 h prior to switching the cells in hypoxic chamber.

Techniques: Permeability, Cell Culture