Journal: Acta Histochemica et Cytochemica

Article Title: Effect of Hepatic Lipid Overload on Accelerated Hepatocyte Proliferation Promoted by HGF Expression via the SphK1/S1PR2 Pathway in MCD-diet Mouse Partial Hepatectomy

doi: 10.1267/ahc.24-00046

Figure Lengend Snippet: SphK1/S1P signaling in mouse liver during regeneration. qPCR analysis of ( A ) SphK1 , ( B ) SphK2 , ( C ) S1PR3 , and ( D ) ABCC1 mRNA expression in the liver of chow- and MCD-diet fed mice after PHx. ( E ) Immunofluorescence analysis of S1PR2 (green) and DAPI (blue) in mouse liver after PHx. Bar = 20 μm; 40× objective lenses were used. ( F ) Quantitative analysis of S1PR2-positive cells in the liver of chow- and MCD-diet fed mice after PHx. Data represent the mean ± SEM for 6–8 mice per group. Asterisks indicate statistically significant differences (*p < 0.05, **p < 0.01 and ***p < 0.001).

Article Snippet: The sections were then reacted with the following primary antibodies for 16–17 hr: anti-proliferating cell nuclear antigen (PCNA) (Dako, M0879), anti-FAT/CD36 (Novus Biologicals, NB400-144), anti-S1PR2 (Novus Biologicals, NBP2-26691), anti-HNF-4α (Abcam, ab181614), anti-F4/80 (Abcam, ab6640), and anti-Ly6G (Biolegend, #127605).

Techniques: Expressing, Immunofluorescence

Journal: Acta Histochemica et Cytochemica

Article Title: Effect of Hepatic Lipid Overload on Accelerated Hepatocyte Proliferation Promoted by HGF Expression via the SphK1/S1PR2 Pathway in MCD-diet Mouse Partial Hepatectomy

doi: 10.1267/ahc.24-00046

Figure Lengend Snippet: Expression of S1PR2 in mouse liver. Double immunofluorescence analysis of S1PR2 (green) and HNF-4α (red staining in upper panel), F4/80 (red staining in middle panel), Ly6G (red staining in lower panel), and DAPI (blue) in the liver of MCD-diet fed mice at 12hr after PHx. Arrows indicate double-positive cells. Bar = 20 μm.

Article Snippet: The sections were then reacted with the following primary antibodies for 16–17 hr: anti-proliferating cell nuclear antigen (PCNA) (Dako, M0879), anti-FAT/CD36 (Novus Biologicals, NB400-144), anti-S1PR2 (Novus Biologicals, NBP2-26691), anti-HNF-4α (Abcam, ab181614), anti-F4/80 (Abcam, ab6640), and anti-Ly6G (Biolegend, #127605).

Techniques: Expressing, Immunofluorescence, Staining

Journal: Acta Histochemica et Cytochemica

Article Title: Effect of Hepatic Lipid Overload on Accelerated Hepatocyte Proliferation Promoted by HGF Expression via the SphK1/S1PR2 Pathway in MCD-diet Mouse Partial Hepatectomy

doi: 10.1267/ahc.24-00046

Figure Lengend Snippet: HGF and FGF2 mRNA expression in S1PR2-positive cells after PHx. ( A ) Macrophages (F4/80) and neutrophils (Ly6G) were isolated from the liver of chow- and MCD-diet fed mice at 0, 6, and 12 hr after PHx. The percentage of macrophages ( B ) and neutrophils ( C ) in the liver of chow- and MCD-diet fed mice. HGF and FGF2 mRNA expression levels in ( D , F ) macrophages and ( E , G ) neutrophils isolated from the liver of chow- and MCD-diet fed mice at 0, 6 and 12 hr after PHx. Data represent the mean ± SEM for 3 mice per group. Asterisks indicate statistically significant differences (*p < 0.05, **p < 0.01 and ***p < 0.001).

Article Snippet: The sections were then reacted with the following primary antibodies for 16–17 hr: anti-proliferating cell nuclear antigen (PCNA) (Dako, M0879), anti-FAT/CD36 (Novus Biologicals, NB400-144), anti-S1PR2 (Novus Biologicals, NBP2-26691), anti-HNF-4α (Abcam, ab181614), anti-F4/80 (Abcam, ab6640), and anti-Ly6G (Biolegend, #127605).

Techniques: Expressing, Isolation

Journal: Pharmacology Research & Perspectives

Article Title: Involvement of S1P 1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells

doi: 10.1002/prp2.68

Figure Lengend Snippet: S1P receptor antagonists attenuate phosphorylation responses of ERK1/2 in HUVEC induced by COA-Cl and S1P. The figure shows the results of immunoblot (IB) analyses in which HUVEC were treated with COA-Cl or S1P either in the presence or absence of pretreatment with antagonists specific for S1P receptors. (A) Effects of the S1P 1 antagonist W146. HUVEC were treated with 10 μ mol/L of W146 for 30 min, followed by 100 μ mol/L of COA-Cl for 15 min. They were then subjected to IB analyses for phospho- and total-ERK1/2. The upper half of the panel shows the representative results of four independent experiments, which yielded equivalent data. The results are summarized in the lower half graphs. (B) VPC23019, a dual antagonist for S1P 1 /S1P 3 (5 μ mol/L for 15 min), was used instead of W146 ( n = 6). (C and D) Cells were treated with 1 μ mol/L of S1P for 5 min instead of COA-Cl ( n = 4), respectively. * P < 0.05 versus vehicle alone. † P < 0.05 versus cells not treated with W146 or VPC23019.

Article Snippet: Rabbit polyclonal anti-S1P 2 antibody was from Alomone (Jerusalem, Israel).

Techniques: Western Blot

Journal: Pharmacology Research & Perspectives

Article Title: Involvement of S1P 1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells

doi: 10.1002/prp2.68

Figure Lengend Snippet: Immunoblot assay and RT-PCR analysis of HUVEC for S1P receptor subtypes and effects of siRNA. (A) Results of immunoblot (IB) analyses using lysates derived from HUVEC and tissue homogenates derived from healthy male rats. Equal quantities of cellular proteins were separated and probed using antibodies directed to S1P receptor subtypes S1P 1–3 or to MAP kinases ERK1/2, as indicated. HUVEC expressed abundant S1P 1 and detectable S1P 3 immunoreactive signals but did not express S1P 2 ( n = 3). (B) Effective knockdown of S1P receptor proteins in HUVEC transiently transfected with siRNA directed to S1P 1 or S1P 3 . In the left half of the figure, the cells were transfected with siRNA specific for S1P 1 (Qiagen Hs_EDG1_1, 40 nmol/L) or negative control siRNA. Forty-eight hours after transfection, the cells were harvested and subjected to a series of IB assays using antibodies directed to S1P 1 , S1P 3 , ERK1/2, VEGFR2, GAPDH and cyclophilin-B (Cy-B). In the right half, the cells were transfected with siRNA specific for S1P 3 (Qiagen Hs_EDG3_6, 20 nmol/L) or control siRNA. For both halves, six independent cultures were tested that yielded similar results. siRNA directed to S1P 1 or S1P 3 leads to significant and specific knockdown of their target protein. (C) Results of RT-PCR assays. HUVEC were transiently transfected with Qiagen siRNA directed to human S1P 1 receptor or with negative control. They were then subjected to RNA isolation and RT-PCR assays using primer pairs as indicated. Note that these cells express detectable levels of S1P receptor subtypes S1P 1−3 along with VEGFR2 and GAPDH. Densitometric analysis revealed that the expression level of S1P 1 transcript relative to GAPDH in cells treated with siRNA specific to S1P 1 decreased to 24.5% ± 5.5% of the control, while those of S1P 3 , S1P 2 , and VEGFR2 remained unchanged ( n = 3).

Article Snippet: Rabbit polyclonal anti-S1P 2 antibody was from Alomone (Jerusalem, Israel).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Transfection, Negative Control, Isolation, Expressing

Journal: Pharmacology Research & Perspectives

Article Title: Involvement of S1P 1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells

doi: 10.1002/prp2.68

Figure Lengend Snippet: Effects of siRNA directed to S1P receptors on HUVEC stimulated with COA-Cl or S1P. The results of immunoblot (IB) analyses using lysates derived from HUVEC treated with COA-Cl or S1P that had been transfected with siRNA directed to S1P receptor subtypes are shown. (A) HUVEC transfected with siRNA specific for S1P 1 or control oligonucleotides were treated with COA-Cl (100 μ mol/L, upper half) for the times indicated, followed by IB analyses for phosphorylated and total forms of ERK1/2. (B) Transfection was performed using siRNA specific for S1P 3 . (C and D) Cells were treated with S1P (100 nmol/L) instead of COA-Cl ( n = 4) for each subset. * P < 0.05 versus vehicle alone. † P < 0.05 versus control siRNA.

Article Snippet: Rabbit polyclonal anti-S1P 2 antibody was from Alomone (Jerusalem, Israel).

Techniques: Western Blot, Derivative Assay, Transfection

Journal: Pharmacology Research & Perspectives

Article Title: Involvement of S1P 1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells

doi: 10.1002/prp2.68

Figure Lengend Snippet: Promotion of tube formation activity by COA-Cl in HUVEC and inhibitory effects of S1P 1 antagonists and a Src tyrosine kinase. The results of tube formation analyses are shown. (A) HUVEC were cocultured with normal human fibroblasts for 10 days in the presence or absence of COA-Cl (100 μ mol/L), with/without W146 (20 μ mol/L) or VPC23019 (5 μ mol/L), as indicated. Endothelial tube formation was then identified as CD31-positive signals in microphotographs, which were subsequently quantified in the graph. The right half of the figure summarizes the results derived from four independent assays, representative images of which are shown in the left half. (B) Certain cells were treated with PP2 (2.5 μ mol/L). In both panels, scale bars correspond to 500 micrometer ( n = 4).

Article Snippet: Rabbit polyclonal anti-S1P 2 antibody was from Alomone (Jerusalem, Israel).

Techniques: Activity Assay, Derivative Assay

Journal: Pharmacology Research & Perspectives

Article Title: Involvement of S1P 1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells

doi: 10.1002/prp2.68

Figure Lengend Snippet: Displacement of [ 3 H]S1P by COA-Cl in receptor ligand-binding competition assay using membrane preparation derived from S1P 1 -overexpressing chem-1 cells. The figure shows the results of receptor ligand-binding competition assay in which [ 3 H]S1P was bound to membrane preparation derived from chem-1 cells overexpressing S1P 1 . Increasing concentrations of COA-Cl, unlabeled S1P, or unlabeled adenosine were included along with [ 3 H]S1P (A−C, respectively). In each panel, 100% and 0% correspond to the [ 3 H]S1P binding values obtained in the presence of the vehicle and excess S1P, respectively ( n = 6−9). (D) Degrees of [ 3 H]S1P binding obtained in the presence of the vehicle, COA-Cl (500 μ mol/L), S1P (10 μ mol/L) and adenosine (2.5 mmol/L). COA-Cl displaced [ 3 H]S1P but adenosine did not.

Article Snippet: Rabbit polyclonal anti-S1P 2 antibody was from Alomone (Jerusalem, Israel).

Techniques: Ligand Binding Assay, Competitive Binding Assay, Derivative Assay, Binding Assay

Journal: Pharmacology Research & Perspectives

Article Title: Involvement of S1P 1 receptor pathway in angiogenic effects of a novel adenosine-like nucleic acid analog COA-Cl in cultured human vascular endothelial cells

doi: 10.1002/prp2.68

Figure Lengend Snippet: Characterization of ERK1/2 responses to COA-Cl in CHO-EDG1 cells. Shown are the results of protein immunoblot (IB) assay of cell lysates derived from CHO-EDG1 cells and their parental CHO-K1 cells. They were treated with increasing concentrations of COA-Cl (A) and S1P (B). They were then subjected to IB with antibodies directed to phosphorylated and total forms of ERK, as indicated. Left side shows representative images and right side shows graphic summaries, where closed and open circles represent values derived from CHO-K1 and CHO-EDG1 cells, respectively ( n = 4). * P < 0.05 and ** P < 0.01 versus vehicle-treated cells. †† P < 0.01 versus CHO-K1 cells.

Article Snippet: Rabbit polyclonal anti-S1P 2 antibody was from Alomone (Jerusalem, Israel).

Techniques: Western Blot, Derivative Assay

Journal: Molecular medicine reports

Article Title: Effects of S1PR2 antagonist on blood pressure and angiogenesis imbalance in preeclampsia rats.

doi: 10.3892/mmr.2021.12095

Figure Lengend Snippet: Figure 1. S1PR2 is increased in the serum and placental tissue of PE rats. (A) Measurement of S1P in serum via ELISA. (B) The expression of S1PR1, S1PR2 and S1PR3 in serum of control and PE rats was analyzed by reverse transcription‑quantitative PCR analysis. (C) The effect of S1P on the expression of S1PR2 in the plasma of PE rats was analyzed via ELISA. (D) The effect of S1P on the expression of S1PR2 in the placental tissues of PE rats was analyzed by western blotting. n=3. *P<0.05, **P<0.01 and ***P<0.001 vs. Control group; #P<0.05 and ###P<0.001 vs. Model group; ∆P<0.05 and ∆∆∆P<0.001 vs. S1P group. S1P, sphingosine‑1‑phosphate; S1PR2, sphingosine‑1‑phosphate receptor 2; PE, preeclampsia; ELISA, enzyme‑linked immunosorbent assay.

Article Snippet: The membranes were incubated with primary antibodies against S1PR2 (cat. no. 21180‐1‐AP; 1:1,000; ProteinTech Group, Inc.), VEGF (cat. no. 66828‐1‐AP; 1:1,000; ProteinTech Group, Inc.), Flt‐1 (cat. no. 13687‐1‐AP; 1:1,000; ProteinTech Group, Inc.), endo‐ thelial (e)NOS (cat. no. ab76198; 1:1,000; Abcam) and β‐actin (cat. no. ab8226; 1:1,000; Abcam) overnight at 4 ̊C.

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Control, Clinical Proteomics, Western Blot

Journal: Molecular medicine reports

Article Title: Effects of S1PR2 antagonist on blood pressure and angiogenesis imbalance in preeclampsia rats.

doi: 10.3892/mmr.2021.12095

Figure Lengend Snippet: Figure 2. Inhibition of S1PR2 with JTE‑013 decreases BP in PE rats. Effect of JTE‑013 on (A) SBP and (B) DBP in PE rats in tail‑cuff measurement. n=3. *P<0.05, **P<0.01 and ***P<0.001 vs. Control group; #P<0.05, ##P<0.01 and ###P<0.001 vs. Model group; ∆P<0.05 vs. Model + S1PR2 antagonist low group. S1PR2, sphingosine‑1‑phosphate receptor 2; PE, preeclampsia; BP, blood pressure; SBP, systolic blood pressure; DBP, diastolic blood pressure.

Article Snippet: The membranes were incubated with primary antibodies against S1PR2 (cat. no. 21180‐1‐AP; 1:1,000; ProteinTech Group, Inc.), VEGF (cat. no. 66828‐1‐AP; 1:1,000; ProteinTech Group, Inc.), Flt‐1 (cat. no. 13687‐1‐AP; 1:1,000; ProteinTech Group, Inc.), endo‐ thelial (e)NOS (cat. no. ab76198; 1:1,000; Abcam) and β‐actin (cat. no. ab8226; 1:1,000; Abcam) overnight at 4 ̊C.

Techniques: Inhibition, Control

Journal: Molecular medicine reports

Article Title: Effects of S1PR2 antagonist on blood pressure and angiogenesis imbalance in preeclampsia rats.

doi: 10.3892/mmr.2021.12095

Figure Lengend Snippet: Figure 3. Effect of JTE‑013 on serum NO and iNOS and the expression of eNOS in placental tissues. (A) Summarized data showing the inhibitory effect of JTE‑013 on serum NO levels in PE rats. (B) Summarized data showing the inhibitory effect of JTE‑013 on serum iNOS levels in PE rats. (C) Summarized data showing that JTE‑013 prevented the decreased expression of eNOS in placental tissues of PE rats. n=3. *P<0.05, **P<0.01 and ***P<0.001 vs. Control group; ##P<0.01 and ###P<0.001 vs. Model group; ∆P<0.05 and ∆∆∆P<0.001 vs. Model + S1PR2 antagonist low group. S1PR2, sphingosine‑1‑phosphate receptor 2; PE, preeclampsia; iNOS, inducible nitric oxide synthase; eNOS, endothelial nitric oxide synthase; NO, nitric oxide.

Article Snippet: The membranes were incubated with primary antibodies against S1PR2 (cat. no. 21180‐1‐AP; 1:1,000; ProteinTech Group, Inc.), VEGF (cat. no. 66828‐1‐AP; 1:1,000; ProteinTech Group, Inc.), Flt‐1 (cat. no. 13687‐1‐AP; 1:1,000; ProteinTech Group, Inc.), endo‐ thelial (e)NOS (cat. no. ab76198; 1:1,000; Abcam) and β‐actin (cat. no. ab8226; 1:1,000; Abcam) overnight at 4 ̊C.

Techniques: Expressing, Control

Journal: Molecular medicine reports

Article Title: Effects of S1PR2 antagonist on blood pressure and angiogenesis imbalance in preeclampsia rats.

doi: 10.3892/mmr.2021.12095

Figure Lengend Snippet: Figure 4. JTE‑013 inhibits the PE model‑induced expression levels of VEGF and Flt‑1 receptor in PE rats. (A) Summarized data showing the inhibitory effect of JTE‑013 on the PE model‑induced mRNA levels of VEGF and Flt‑1 in reverse transcription‑quantitative PCR assay. (B) Representative western blotting images and summarized data showing the inhibitory effect of JTE‑013 on the PE model‑induced protein levels of VEGF and Flt‑1 in the placental tissues of PE rats. n=3. *P<0.05 and ***P<0.001 vs. Control group; #P<0.05, ##P<0.01 and ###P<0.001 vs. Model group; ∆∆P<0.01 and ∆∆∆P<0.001 vs. Model + S1PR2 antagonist low group. S1PR2, sphingosine‑1‑phosphate receptor 2; PE, preeclampsia; VEGF, vascular endothelial growth factor; Flt‑1, fms‑like tyrosine kinase 1.

Article Snippet: The membranes were incubated with primary antibodies against S1PR2 (cat. no. 21180‐1‐AP; 1:1,000; ProteinTech Group, Inc.), VEGF (cat. no. 66828‐1‐AP; 1:1,000; ProteinTech Group, Inc.), Flt‐1 (cat. no. 13687‐1‐AP; 1:1,000; ProteinTech Group, Inc.), endo‐ thelial (e)NOS (cat. no. ab76198; 1:1,000; Abcam) and β‐actin (cat. no. ab8226; 1:1,000; Abcam) overnight at 4 ̊C.

Techniques: Expressing, Western Blot, Control

Journal: Molecular medicine reports

Article Title: Effects of S1PR2 antagonist on blood pressure and angiogenesis imbalance in preeclampsia rats.

doi: 10.3892/mmr.2021.12095

Figure Lengend Snippet: Figure 5. JTE‑013 attenuates pathological changes in placental tissues and decreases inflammation in PE rats. (A) Summarized data showing the inhibitory effect of JTE‑013 on the increased expression of serum TNF‑α, IL‑1β and IL‑6, as determined by an enzyme‑linked immunosorbent assay. (B) Summarized data showing JTE‑013 attenuated the infiltration of inflammatory cells in placental tissues, as detected by hematoxylin and eosin staining (magnification, x100 or 200). n=3. *P<0.05, **P<0.01 and ***P<0.001 vs. Control group; ##P<0.01 and ###P<0.001 vs. Model group; ∆P<0.05 and ∆∆P<0.01 vs. Model + S1PR2 antagonist low group. PE, preeclampsia; TNF‑α, tumor necrosis factor‑α; IL‑, interleukin; S1PR2, sphingosine‑1‑phosphate receptor 2; lab, labyrinth; JZ, junctional zone; de, decidua.

Article Snippet: The membranes were incubated with primary antibodies against S1PR2 (cat. no. 21180‐1‐AP; 1:1,000; ProteinTech Group, Inc.), VEGF (cat. no. 66828‐1‐AP; 1:1,000; ProteinTech Group, Inc.), Flt‐1 (cat. no. 13687‐1‐AP; 1:1,000; ProteinTech Group, Inc.), endo‐ thelial (e)NOS (cat. no. ab76198; 1:1,000; Abcam) and β‐actin (cat. no. ab8226; 1:1,000; Abcam) overnight at 4 ̊C.

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

Journal: Frontiers in Cell and Developmental Biology

Article Title: MiR-9 Promotes Angiogenesis via Targeting on Sphingosine-1- Phosphate Receptor 1

doi: 10.3389/fcell.2020.00755

Figure Lengend Snippet: Expression analysis of miR-9 and S1P receptors in tumor ECs and normal ECs basing on EndoDB databases. (A) Gene expression heatmap of normal ECs (NEC) vs. tumor ECs (TEC). Red indicates high-expression levels, and blue indicates low-expression levels. (B) Expression of the miR-9-1, miR-9-2, and miR-9-3 in NECs and TECs. MUS: musculus; Homo: Homo sapiens. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: After determination of protein concentration using a protein determination kit (Cayman Chemical Company, Ann Arbor, MI, United States), equal amounts (20–30 μg) of protein samples were size-fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electrotransferred onto a polyvinylidene fluoride membrane (Millipore, United States), blocked with 5% non-fat milk in PBS, and hybridized with antibodies against S1P 1 (TA311878; Origene, Rockville, MD, United States), S1P 2 (TA311287; Origene), and S1P 3 (TA321693; Origene) at 4°C overnight.

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: MiR-9 Promotes Angiogenesis via Targeting on Sphingosine-1- Phosphate Receptor 1

doi: 10.3389/fcell.2020.00755

Figure Lengend Snippet: Relationship between miR-9 and S1P receptors. The correlation between the significant differentially expressed miR-9 loci and specific S1P receptors in normal ECs (NEC) and tumor ECs (TEC) were performed. (A) Correlation between miR-9-2 and S1P receptors in Wnt-medulloblastoma; (B) correlation between miR-9-1, miR-9-2, and S1P 2 in Shh-medulloblastoma. Then, the levels of S1P receptors in HUVECs upon miR-9 mimics transfection were detected by qRT-PCR. (C) miR-9 levels in HUVECs overexpressing miR-9 (left) and mRNA expression of S1P 1 , S1P 2 , and S1P 3 (right). NC: miR-9 mimics negative control. Mean ± SEM. n = 4, * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: After determination of protein concentration using a protein determination kit (Cayman Chemical Company, Ann Arbor, MI, United States), equal amounts (20–30 μg) of protein samples were size-fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electrotransferred onto a polyvinylidene fluoride membrane (Millipore, United States), blocked with 5% non-fat milk in PBS, and hybridized with antibodies against S1P 1 (TA311878; Origene, Rockville, MD, United States), S1P 2 (TA311287; Origene), and S1P 3 (TA321693; Origene) at 4°C overnight.

Techniques: Transfection, Quantitative RT-PCR, Expressing, Negative Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: MiR-9 Promotes Angiogenesis via Targeting on Sphingosine-1- Phosphate Receptor 1

doi: 10.3389/fcell.2020.00755

Figure Lengend Snippet: Western blot detection of S1P 1 , S1P 2 , and S1P 3 in HUVECs overexpressing miR-9. Cell lysates were collected from HUVECs with or without NC or miR-9 transfection, and then 20 μg of protein samples were subjected to western blot analysis. (A) Western blot of cell lysates using anti-S1P1, anti-S1P2, and anti-S1P3. (B) The quantification was performed with ImageJ. The protein expression was normalized to the GAPDH level. NC: miR-9 mimics negative control. Mean ± SEM. n = 3, *** P < 0.001.

Article Snippet: After determination of protein concentration using a protein determination kit (Cayman Chemical Company, Ann Arbor, MI, United States), equal amounts (20–30 μg) of protein samples were size-fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electrotransferred onto a polyvinylidene fluoride membrane (Millipore, United States), blocked with 5% non-fat milk in PBS, and hybridized with antibodies against S1P 1 (TA311878; Origene, Rockville, MD, United States), S1P 2 (TA311287; Origene), and S1P 3 (TA321693; Origene) at 4°C overnight.

Techniques: Western Blot, Transfection, Expressing, Negative Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: MiR-9 Promotes Angiogenesis via Targeting on Sphingosine-1- Phosphate Receptor 1

doi: 10.3389/fcell.2020.00755

Figure Lengend Snippet: Associations between S1P 1 , S1P 3 , and miR-9 were detected by dual-luciferase reporter assay. The structures of S1P 1 mRNA (transcript variant 1 M_001400.5, and transcript variant 2 NM_001320730.1; the variant 2 differs in the 5′UTR compared to variant 1, both variants 1 and 2 encode the same protein) and S1P 3 mRNA (NM_005226.4) including coding DNA sequence (CDS) and untranslated regions were depicted. The blue band indicated the putative miR-9 binding sequence in the target mRNA molecules. The binding sites located at 3′UTR were screened for Dual-Luciferase Reporter Assay analysis. (A) Putative wild-type (WT) binding sequence and its mutation (MUT) sequence in the 3′UTR of S1P 1 . Luciferase activity was measured. (B) Putative WT binding sequence and its MUT sequence in the 3′UTR of S1P 3 . Luciferase activity was measured. NC: miR-9 mimics negative control. Mean ± SEM. n = 3, *** P < 0.001.

Article Snippet: After determination of protein concentration using a protein determination kit (Cayman Chemical Company, Ann Arbor, MI, United States), equal amounts (20–30 μg) of protein samples were size-fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electrotransferred onto a polyvinylidene fluoride membrane (Millipore, United States), blocked with 5% non-fat milk in PBS, and hybridized with antibodies against S1P 1 (TA311878; Origene, Rockville, MD, United States), S1P 2 (TA311287; Origene), and S1P 3 (TA321693; Origene) at 4°C overnight.

Techniques: Luciferase, Reporter Assay, Variant Assay, Sequencing, Binding Assay, Mutagenesis, Activity Assay, Negative Control

Journal: Frontiers in Cell and Developmental Biology

Article Title: MiR-9 Promotes Angiogenesis via Targeting on Sphingosine-1- Phosphate Receptor 1

doi: 10.3389/fcell.2020.00755

Figure Lengend Snippet: Effect of S1P 1 on angiogenesis. Cells were transfected with pcDNA3.1 vector expressing S1P 1 (+S1P 1 ) or pcDNA3.1 vector (vector). For the rescue assay, cells overexpressing miR-9 were transfected with pcDNA3.1 vector expressing S1P 1 (miR-9 + S1P 1 ) or pcDNA3.1 vector (miR-9 + vector). After transfection for 48 h, the protein level, migration, invasion and in vitro angiogenesis were performed. (A) Western blot of S1P 1 and quantification. Mean ± SEM. n = 3, * P < 0.05, *** P < 0.001. ### P < 0.001. (B–D) Transwell assays (B) of migration (top) and invasion (bottom) of HUVECs overexpressing S1P 1 in response to 50 ng/mL hVEGF. Mean ± SEM, n = 4. *** P < 0.001. (E–G) Tube formation (E) , normalized tube length (F) , and number of junctions (J) formed by HUVECs overexpressing S1P 1 in response to 50 ng/mL hVEGF. Mean ± SEM, n = 4. * P < 0.05, ** P < 0.01. (H–J) Transwell assays (H) of migration (top) and invasion (bottom) of HUVECs overexpressing miR-9 and S1P 1 . NC: miR-9 mimics negative control. Mean ± SEM, n = 4. * P < 0.05, ** P < 0.01, *** P < 0.001; ### P < 0.001. (K–M) Tube formation (K) , normalized tube length (L) , and the number of junctions (M) formed by HUVECs overexpressing miR-9 and S1P 1 . NC: miR-9 mimics negative control. Mean ± SEM, n = 4. * P < 0.05; ## P < 0.01.

Article Snippet: After determination of protein concentration using a protein determination kit (Cayman Chemical Company, Ann Arbor, MI, United States), equal amounts (20–30 μg) of protein samples were size-fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electrotransferred onto a polyvinylidene fluoride membrane (Millipore, United States), blocked with 5% non-fat milk in PBS, and hybridized with antibodies against S1P 1 (TA311878; Origene, Rockville, MD, United States), S1P 2 (TA311287; Origene), and S1P 3 (TA321693; Origene) at 4°C overnight.

Techniques: Transfection, Plasmid Preparation, Expressing, Rescue Assay, Migration, In Vitro, Western Blot, Negative Control

Journal: Cell reports

Article Title: Crosstalk between pro-survival sphingolipid metabolism and complement signaling induces inflammasome-mediated tumor metastasis

doi: 10.1016/j.celrep.2022.111742

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: S1P2/EDG-5/S1PR2 Antibody , Novus Biologicals , Cat# NBP2-26691.

Techniques: Recombinant, Membrane, Wound Healing Assay, Invasion Assay, MTT Cell Proliferation, Protease Inhibitor, Western Blot, Lysis, In Situ Hybridization, Plasmid Preparation, cDNA Synthesis, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, RNAscope, RNA Sequencing, Gene Expression, Knock-Out, shRNA, CRISPR, Cloning, Software, Imaging

Journal: The Journal of Biological Chemistry

Article Title: TGF-β/SMAD3 Pathway Stimulates Sphingosine-1 Phosphate Receptor 3 Expression

doi: 10.1074/jbc.M116.740084

Figure Lengend Snippet: Up-regulation of S1PR3 in human lung adenocarcinomas. A, qPCR quantitation of S1PR3 mRNA in cDNA arrays of human lung adenocarcinoma specimens (OriGene, HLRT101 and HLRT105). **, p < 0.01, Student's t test. B, qPCR quantitation of S1PR2 mRNA in a cDNA array of human lung cancers (OriGene, HLRT105). **, p < 0.01, Student's t test. C, HEK293 cells were transfected with S1PR3 or pcDNA vector. Transfected cells were immunostained with anti-S1PR3 (Cayman Chemical) (IMF, left panels). Arrows, nonspecific fluorescent precipitates used for image orientation. Scale bar = 33 μm. D, anti-S1PR3 staining of human lung adenocarcinoma tumor microarray (Accumax 306). AdC, adenocarcinoma; N, adjacent normal lung tissue. E, immunostaining intensity was quantitated with the National Institutes of Health ImageJ software. Data, analyzed with GraphPad Prism 5 software, are shown as mean ± S.E. Statistical significance was analyzed by Student's t test. F, representative images of anti-S1PR3 staining of human lung adenocarcinoma and the respective adjacent normal lung epithelial tissue. G, quantitation of anti-S1PR3 staining of human lung squamous carcinoma microarray (Accumax 306). Data are mean ± S.E. Statistical significance was analyzed by Student's t test. H, representative images of anti-S1PR3 staining of human lung squamous carcinoma and the respective adjacent normal lung epithelial tissue.

Article Snippet: B , qPCR quantitation of S1PR2 mRNA in a cDNA array of human lung cancers (OriGene, HLRT105).

Techniques: Quantitation Assay, Transfection, Plasmid Preparation, Staining, Microarray, Immunostaining, Software

Journal: The Journal of Biological Chemistry

Article Title: TGF-β/SMAD3 Pathway Stimulates Sphingosine-1 Phosphate Receptor 3 Expression

doi: 10.1074/jbc.M116.740084

Figure Lengend Snippet: TGF-β/SMAD3 signaling axis up-regulates S1PR3. A, HBEC2-KT cells were treated with TGF-β (1 ng/ml) for various times. mRNA levels of S1P receptors were measured by qPCR analysis. Data are mean ± S.D. of triplicate determinations. *, p < 0.05, Student's t test. B, protein levels of S1PR3 in TGF-β (1 ng/ml)-treated HBEC2-KT cells. Lower panel, Western blot intensity was quantitated by National Institutes of Health ImageJ. Data (normalized to actin) are mean ± S.D. of triplicate determinations. * and **, p < 0.05 and 0.01, respectively, Student's t test. C, CHO cells were transduced with adenoviral particles (multiplicity of infection of 200) carrying S1PR1, S1PR2, or S1PR3 vector for 20 h as we described (8). Extracts were blotted with antibody against S1PR3 (Cayman), S1PR2 (Cayman), or S1PR1 (E49) (8). D, mRNAs of S1PR3 and TGF-β in minced C57BL/6 mouse lungs (∼1–2 mm3) infected with adenoviral active TGF-β (Ad-TGF-β, 1 × 108 pfu/ml) or empty vector (Ad-Ctrl) (37 °C, 24 h). **, p < 0.01, n = 5, Student's t test. E, mRNA levels of SphK1 and SphK2 in TGF-β-treated HBEC2-KT cells. F, HBEC2-KT cells (2 × 106 cells in 100-mm dish, 10 ml of cultural medium) were treated with TGF-β (1 ng/ml) for 24 h. Medium was quantitated for S1P, ceramide (Cer), and sphingomyelin (SPM) by LC-MS/MS (29, 46). G, HBEC2-KT were pretreated for 30 min with inhibitors. S1PR3 levels were measured by qPCR, following TGF-β treatment (4 h). The following inhibitors were used: SB4, TGF-β receptor I (SB-431542, 10 μm); SIS3, SMAD3 (2 μm); SB2, p38 kinase (SB-203580, 50 nm); BAY, NFκB (BAY11-7085, 10 μm); JII, JNK (JNK inhibitor II, 10 μm). *, p < 0.05; dashed line, non-statistical significance; n = 3, ANOVA. Each experiment was repeated 2–3 times with similar results. H, cells were pretreated for 30 min with inhibitors, followed by stimulation with TGF-β (1 ng/ml). Activation of p38, JNK, and NFκB was measured by Western blotting with phospho-p38 (P-p38), phospho-JNK (P-p54JNK and P-p46JNK), and phospho-IκBα (p-IκBα). Inhibitors used are: SB-203580 (50 nm) for p38 kinase, JNK inhibitor II (10 μm) for JNK, and BAY11-7085 (10 μm) for NFκB.

Article Snippet: B , qPCR quantitation of S1PR2 mRNA in a cDNA array of human lung cancers (OriGene, HLRT105).

Techniques: Western Blot, Transduction, Infection, Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: TGF-β/SMAD3 Pathway Stimulates Sphingosine-1 Phosphate Receptor 3 Expression

doi: 10.1074/jbc.M116.740084

Figure Lengend Snippet: S1PR3 regulates growth and lung colonization of lung adenocarcinoma cells. A, H1793 cells were stably transfected with sh-S1PR3 or pRS (sh-Ctrl) vector (11, 16). mRNA levels of S1PR3 were quantitated with qPCR analysis. B, H1793 cells (1 × 106 cells), stably transfected with sh-S1PR3 or sh-Ctrl vector, were subcutaneously inoculated in Scid mice. Tumor volume was measured in two dimensions using calipers, and volume was determined using the formula width2 × length × 0.52 (49). C, 4 weeks after inoculation, tumors were removed and weighed. D, Scid mice were injected with H1793 cells transfected with sh-S1PR3 or sh-Ctrl vector (1 × 106 cells) via tail vein route. 28 days later, tumor nodules on lung surface were scored. E, representative images of lung injected with H1793-sh-Ctrl and H1793-sh-S1PR3 cells. Arrows, tumor nodules. Scale bar = 0.5 cm. F, volume of xenograft tumors in athymic nude mice subcutaneously implanted with H1299 cells stably transfected with S1PR3 or control pcDNA vector (1 × 106 cells) (11, 16). G, qPCR quantitation of S1PR3 levels in H1299/pcDNA and H1299/S1PR3 cells. **, p < 0.01, n = 6, ANOVA.

Article Snippet: B , qPCR quantitation of S1PR2 mRNA in a cDNA array of human lung cancers (OriGene, HLRT105).

Techniques: Stable Transfection, Transfection, Plasmid Preparation, Injection, Quantitation Assay