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: Journal of neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance.

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: Fig. 1 HFD feeding induces insulin resistance and disruptes BCAAs catabolism in APP/PS1 mice. A Body weight of APP/PS1 mice during experiment period (n = 11 to 12). B Blood glucose levels in the glucose tolerance test (OGTT) and the area under the curve (AUC) of APP/ PS1 mice (n = 8). C Fasting serum glucose and insulin levels, and corresponding HOMA-IR index of APP/PS1 mice (n = 8). D Heatmaps of the amino acid levels in serum of APP/PS1 mice in the indicated groups (n = 8). E Comparison of the levels of BCAAs and BCKAs in cerebral cortex (n = 8). F Heatmap analysis of the Spearman correlation of circulating and cortical levels of BCAAs and BCKAs. Western blot analysis of the levels of BCAAs catabolism-related protein in liver (G), skeletal muscle (H), and cerebral cortex (I), and the corresponding quantification results (n = 4–5). Data are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, two-way or one-way ANOVA, followed by Tukey’s multiple comparisons test

Article Snippet: Unless indicated otherwise, BV2 cells were treated with vehicle, BCAAs (5 mM, leucine: valine: isoleucine = 1:1:1) or BCKAs (5 mM, KICA:KMVA:KIVA = 1:1:1) in the presence of Aβ (1 μM) with or without 40 μM sphingosine-1-phosphate (S1P; MedChemExpress) for 4 h. In addition, BV2 cells were treated with vehicle, BCAAs or BCKAs in the presence of Aβ with or without the agents rapamycin (50 nM), chloroquine (40 μM) or 4-octyl itaconate (40 μM) (MedChemExpress) for 12 h.

Techniques: Comparison, Western Blot

Journal: Journal of neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance.

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: Fig. 2 BCAAs addition and restriction affect the development of cognitive deficits and AD-related pathology in HFD-fed APP/PS1 mice. A Escape latency to the platform during the training trails in a Morris water maze (n = 11 to 12). B Target (platform) entries in the probe test (n = 11 to 12). C Target quadrant entries in the probe test (n = 11 to 12). D Mean swimming speed of mice (n = 11 to 12). E Representative swimming tracks of mice in the probe test (n = 11 to 12). (F) Heatmap analysis of the Spearman correlation of cognitive function-related indexes and cortical levels of BCAAs and BCKAs (n = 8). G Western blot analysis of the protein levels of postsynaptic (PSD95) and presynaptic (synaptophysin) markers in cerebral cortex of mice, and the corresponding quantification results (n = 4). (H) Western blot analysis of the protein levels of Aβ secretase (BACE1) in cerebral cortex and the corresponding quantification results (n = 4). I Immunostainings of Aβ plaques by using specific antibody (6E10) in cortex and hippocampus (Scale bars, 200 μm), and quantitation of positive area rate (n = 3). Data are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, two-way or one-way ANOVA, followed by Tukey’s multiple comparisons test

Article Snippet: Unless indicated otherwise, BV2 cells were treated with vehicle, BCAAs (5 mM, leucine: valine: isoleucine = 1:1:1) or BCKAs (5 mM, KICA:KMVA:KIVA = 1:1:1) in the presence of Aβ (1 μM) with or without 40 μM sphingosine-1-phosphate (S1P; MedChemExpress) for 4 h. In addition, BV2 cells were treated with vehicle, BCAAs or BCKAs in the presence of Aβ with or without the agents rapamycin (50 nM), chloroquine (40 μM) or 4-octyl itaconate (40 μM) (MedChemExpress) for 12 h.

Techniques: Western Blot, Quantitation Assay

Journal: Journal of neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance.

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: Fig. 3 BCAAs and BCKAs contribute to microglial phagocytosis deficiency in HFD-fed APP/PS1 mice and BV2 cell. A Representative images of microglia (IBA1) staining in cortex and hippocampus of APP/PS1 mice (Scale bars, 100 μm), and corresponding quantitation of positive cell number (n = 3). B Western blot analysis of IBA1 levels in cortex, and the corresponding quantification results (n = 4). C Representative images of Aβ plaques (6E10) and microglia (IBA1) co-staining in cortex and hippocampus of APP/PS1 mice (Scale bars, 20 μm), and corresponding quantitation of positive cell number around plaque (n = 3). Microglial phagocytosis D and degradation E of fluorescein labeled-Aβ for the indicated time in BV2 cells in the presence or absence of BCAAs/ BCKAs (n = 3). Data are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test

Article Snippet: Unless indicated otherwise, BV2 cells were treated with vehicle, BCAAs (5 mM, leucine: valine: isoleucine = 1:1:1) or BCKAs (5 mM, KICA:KMVA:KIVA = 1:1:1) in the presence of Aβ (1 μM) with or without 40 μM sphingosine-1-phosphate (S1P; MedChemExpress) for 4 h. In addition, BV2 cells were treated with vehicle, BCAAs or BCKAs in the presence of Aβ with or without the agents rapamycin (50 nM), chloroquine (40 μM) or 4-octyl itaconate (40 μM) (MedChemExpress) for 12 h.

Techniques: Staining, Quantitation Assay, Western Blot, Labeling

Journal: Journal of neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance.

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: Fig. 4 BCAAs and BCKAs impact the response of microglial TREM2 and autophagy to Aβ. A Western blot analysis of TREM2-related protein levels in cortex of APP/PS1 mice and the corresponding quantification results (n = 4). B Relative mRNA levels of DAP12 and CD68 in cortex (n = 5). C Western blot analysis of autophagy-related protein levels in cortex and the corresponding quantification results (n = 4). D Relative mRNA levels of BECLIN1 and LAMP1 in cortex (n = 5). Western blot analysis of TREM2-related E and autophagy-related F Protein levels in BV2 cell treated with or without BCAAs/ BCKAs for 4 h in the presence of Aβ and the corresponding quantification results (n = 3). Western blot analysis of TREM2-related G and autophagy-related H protein levels in BV2 cell treated with or without BCAAs/ BCKAs for 12 h in the presence of Aβ and the corresponding quantification results (n = 3). Data are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test

Article Snippet: Unless indicated otherwise, BV2 cells were treated with vehicle, BCAAs (5 mM, leucine: valine: isoleucine = 1:1:1) or BCKAs (5 mM, KICA:KMVA:KIVA = 1:1:1) in the presence of Aβ (1 μM) with or without 40 μM sphingosine-1-phosphate (S1P; MedChemExpress) for 4 h. In addition, BV2 cells were treated with vehicle, BCAAs or BCKAs in the presence of Aβ with or without the agents rapamycin (50 nM), chloroquine (40 μM) or 4-octyl itaconate (40 μM) (MedChemExpress) for 12 h.

Techniques: Western Blot

Journal: Journal of neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance.

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: Fig. 5 BCAAs and BCKAs suppress TREM2 activation through binding to TREM2. A Molecular docking results of L-Leucine and KICA binding to TREM2 extracellular domain. B Biolayer interferometry (BLI) analysis of interaction of BCAAs or BCKAs with TREM2. BCAAs and BCKAs was titrated from 15.63 to 500 μM and 31.25 to 500 μM, respectively. C LC–MS/MS spectra of TREM2 immunoprecipitated L-Leucine-13C of BV2 cells after BCAAs isotope treatment for 1 h. D Thermal stabilization of TREM2 protein evaluated by cellular thermal shift assay (CETSA) after treating BV2 cells with BCAAs/ BCKAs for 4 h (n = 3). E Representative images of binding of fluorescein labeled-Aβ to cell surface TREM2 in BV2 cells after treatment of BCAAs/ BCKAs for 2 h (n = 3, Scale bars, 40 μm). F Representative images of microglial phagocytosis of fluorescein labeled-Aβ in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of S1P for 4 h (n = 3, Scale bars, 40 μm). G Western blot analysis of phosphorylation levels of SYK in BV2 cell after treatment of BCAAs/ BCKAs in the presence or absence of sphingosine-1-phosphate (S1P) for 4 h (n = 3). Data in D, E, F and G are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test

Article Snippet: Unless indicated otherwise, BV2 cells were treated with vehicle, BCAAs (5 mM, leucine: valine: isoleucine = 1:1:1) or BCKAs (5 mM, KICA:KMVA:KIVA = 1:1:1) in the presence of Aβ (1 μM) with or without 40 μM sphingosine-1-phosphate (S1P; MedChemExpress) for 4 h. In addition, BV2 cells were treated with vehicle, BCAAs or BCKAs in the presence of Aβ with or without the agents rapamycin (50 nM), chloroquine (40 μM) or 4-octyl itaconate (40 μM) (MedChemExpress) for 12 h.

Techniques: Activation Assay, Binding Assay, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Thermal Shift Assay, Labeling, Western Blot, Phospho-proteomics

Journal: Journal of neuroinflammation

Article Title: Accumulated BCAAs and BCKAs contribute to the HFD-induced deterioration of Alzheimer's disease via a dysfunctional TREM2-related reduction in microglial β-amyloid clearance.

doi: 10.1186/s12974-024-03314-1

Figure Lengend Snippet: Fig. 6 BCAAs and BCKAs reduce the expression and recycling of microglial TREM2 via inhibiting autophagy. Western blot analysis of TREM2-related A and autophagy-related B protein levels in BV2 cell after treatment of BCAAs/ BCKAs in the presence or absence of rapamycin for 12 h (n = 3). C Representative images of microglial phagocytosis of fluorescein labeled-Aβ in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of rapamycin for 12 h (n = 3, Scale bars, 40 μm). D Representative images of TREM2 recycling in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of rapamycin for 12 h (n = 3, Scale bars, 40 μm). Western blot analysis of TREM2-related E and autophagy-related F protein levels in BV2 cell after treatment of BCAAs/ BCKAs in the presence or absence of chloroquine for 12 h (n = 3). G Representative images of TREM2 recycling in BV2 cells after treatment of BCAAs/ BCKAs in the presence or absence of chloroquine for 12 h (n = 3, Scale bars, 40 μm). Data are means ± SEM. *P < 0.05,**P < 0.01 and ***P < 0.001, one-way ANOVA, followed by Tukey’s multiple comparisons test or Student’s t test

Article Snippet: Unless indicated otherwise, BV2 cells were treated with vehicle, BCAAs (5 mM, leucine: valine: isoleucine = 1:1:1) or BCKAs (5 mM, KICA:KMVA:KIVA = 1:1:1) in the presence of Aβ (1 μM) with or without 40 μM sphingosine-1-phosphate (S1P; MedChemExpress) for 4 h. In addition, BV2 cells were treated with vehicle, BCAAs or BCKAs in the presence of Aβ with or without the agents rapamycin (50 nM), chloroquine (40 μM) or 4-octyl itaconate (40 μM) (MedChemExpress) for 12 h.

Techniques: Expressing, Western Blot, Labeling

Journal: Cell

Article Title: ER Stress Drives Lipogenesis and Steatohepatitis via Caspase-2 Activation of S1P

doi: 10.1016/j.cell.2018.08.020

Figure Lengend Snippet: A) Indicated expression vectors were transfected into HEK293 cells. After 48 hr, cells were incubated with 25 μg/ml N-acetyl-leucine-leucine-norleucinal (ALLN) for 3 hr before harvest. WCL were IB’d with antibodies to SREBP1 and 2. B) Indicated expression vectors were transfected into SCAP-ablated HEK293 cells (293ΔSCAP). After 5 hr, cells were incubated with 1% lipoprotein-deficient serum (LPDS) for 16 hr, followed by ethanol or 50 μM mevalonate + 5 μg/ml cholesterol for 12 hr and 3 hr with ALLN before harvest. WCL, membranes, and NE were IB’d. Red stars: N-terminal Myc epitope-containing S1P polypeptides. C) Intracellular localization of Casp2-HA, S1P-Myc and Flag-SREBP2 in S1P-ablated HEK293 cells (293ΔS1P). Transiently expressed Casp2, S1P, and SREBP2 were visualized by immunofluorescence with HA, Myc, and FLAG antibodies, respectively. Nuclei were counterstained with DAPI. Scale bar: 10 μm. D) Indicated proteins were expressed in HEK293 cells. After 48 hr, WCL and culture supernatants (CS) were IB’d with Myc and S1P antibodies (2 lanes per condition). E) Indicated proteins were expressed in 293ΔSCAP cells. 5 hr after transfection, the cells were incubated with 1% LPDS for 16 hr followed by 3 hr with ALLN. WCL, membranes, and NE were prepared and Casp2 and S1P were IB’d with HA and Myc antibodies. CS were IB’d for S1P. Arrow: cleaved Casp2, red stars: N-terminal S1P fragments that retained the Myc epitope. F) ER and Golgi fractions were isolated by differential centrifugation from livers of LFD-fed 7-week-old MUP-uPA and Casp2−/−/MUP-uPA mice. Proteins in each fraction were de-glycosylated with PNGase F and IB’d as indicated. Star: nonspecific band present in the Golgi fraction of Casp2-null liver, F.L: full-length, C2-Cl.: Casp2-cleaved, A-Cl.: autocleaved. G) BL6 hepatocytes introduced with Adv carrying GFP or uPA. After 24 hr, ER, Golgi and nuclear fractions were isolated by differential centrifugation and IB’d for indicated proteins. F.L: full-length, Cl.: cleaved, L.E: long-exposure, S.E: short-exposure. H) IB of S1P in sera of HFD-fed (12 weeks) mice (n = 3) of indicated genotypes. I) Sera from normal individuals, NAFLD and NASH patients (n = 3 per group) with different liver fibrosis scores were IB’d with S1P antibody.

Article Snippet: Mouse S1P (MC204593) and mouse Casp2 cDNA clone (MC206011) were purchased from Origene Technologies, Inc. Flag-tagged SREBP2 (#32018) cDNA clone was from Addgene.

Techniques: Expressing, Transfection, Incubation, Immunofluorescence, Isolation, Centrifugation

Journal: Neural Plasticity

Article Title: Activation of Sphingosine 1-Phosphate Receptor 1 Enhances Hippocampus Neurogenesis in a Rat Model of Traumatic Brain Injury: An Involvement of MEK/Erk Signaling Pathway

doi: 10.1155/2016/8072156

Figure Lengend Snippet: Hippocampal sphingosine 1-phosphate receptor 1 (S1PR1) expression increased markedly after TBI. (a) The level of S1PR1 protein in hippocampus was detected by western blotting (WB) at 12 hours, 1, 3, 7, 14, 21, and 28 days after trauma. (b) Quantitative analysis indicated that upregulated expression of hippocampal S1PR1 occurred from 12 hours, peaked at 7 days, and remained at a relatively higher level for at least 28 days after TBI ( n = 3, sham group; n = 5 each time-point group of TBI). Further analysis revealed that there was a statistical difference between any two time-point groups after trauma. ∗ P < 0.05 versus sham group; # P < 0.05 versus other time-point groups of TBI.

Article Snippet: Following blocked by 5% skim milk in Tris-buffered saline solution containing 0.1% Tween-20 (TBST), the membranes were incubated overnight at 4°C with the following primary antibodies: rabbit anti-rat S1PR1 antibody (1 : 500, Prosci, 4809, Poway, CA, USA), rabbit anti-rat phosphate Erk (pErk) antibody (1 : 1000, Cell Signaling, 9101, Beverly, MA, USA), rabbit anti-rat total Erk (tErk) antibody (1 : 1000, Cell Signaling, 9102, Beverly, MA, USA), rabbit anti-rat phosphate MEK (pMEK) antibody (1 : 1000, Cell Signaling, 9154, Beverly, MA, USA), and rabbit anti-rat total MEK (tMEK) antibody (1 : 1000, Cell Signaling, 9126, Beverly, MA, USA).

Techniques: Expressing, Western Blot

Journal: Neural Plasticity

Article Title: Activation of Sphingosine 1-Phosphate Receptor 1 Enhances Hippocampus Neurogenesis in a Rat Model of Traumatic Brain Injury: An Involvement of MEK/Erk Signaling Pathway

doi: 10.1155/2016/8072156

Figure Lengend Snippet: S1PR1 activation enhanced NSCs proliferation and neuronal differentiation in hippocampal dentate gyrus (DG) after TBI. (a) Coronal diagram of rat hippocampus, the subgranular zone, granular cells layer, and molecular layer of DG were, respectively, marked by SGZ, GCL, and MOL, the blue pane representing one of microscopic visual fields for counting immunolabeled cells of immunofluorescence (IF). (b) NSCs proliferation in SGZ at 7 days after TBI was assessed by BrdU/SOX2 double-labeling IF. Quantitation analysis showed that, relative to sham group ( n = 3), brain injury induced more double-positive cells in TBI+Vehicle group ( n = 6). Following the treatment of SEW2871, the number of BrdU + /SOX2 + cells further increased in TBI+SEW group ( n = 6). Reversely, administration of VPC23019 resulted in a significant reduction of BrdU + /SOX2 + cells in TBI+SEW+VPC group ( n = 6). (c) Neuronal differentiation of NSCs in SGZ at 28 days after TBI was assessed by BrdU/NeuN double-labeling IF. Statistical data indicated that BrdU + /NeuN + cells increased in TBI+Vehicle group ( n = 6) compared with sham group ( n = 3). And the double-positive cells increased at even higher level after SEW2871 treatment in TBI+SEW group ( n = 6). However, VPC23019 administration caused an evident decrease of BrdU + /NeuN + cells in TBI+SEW+VPC group ( n = 6). (d) and (e) Representative IF microphotographs of hippocampus immunolabeled with BrdU/SOX2 and BrdU/NeuN in all the above four groups at 7 days and 28 days after trauma. Scale bar: 50 μ m. ∗ P < 0.05 versus sham group; # P < 0.05 versus TBI+Vehicle group or TBI+SEW+VPC group.

Article Snippet: Following blocked by 5% skim milk in Tris-buffered saline solution containing 0.1% Tween-20 (TBST), the membranes were incubated overnight at 4°C with the following primary antibodies: rabbit anti-rat S1PR1 antibody (1 : 500, Prosci, 4809, Poway, CA, USA), rabbit anti-rat phosphate Erk (pErk) antibody (1 : 1000, Cell Signaling, 9101, Beverly, MA, USA), rabbit anti-rat total Erk (tErk) antibody (1 : 1000, Cell Signaling, 9102, Beverly, MA, USA), rabbit anti-rat phosphate MEK (pMEK) antibody (1 : 1000, Cell Signaling, 9154, Beverly, MA, USA), and rabbit anti-rat total MEK (tMEK) antibody (1 : 1000, Cell Signaling, 9126, Beverly, MA, USA).

Techniques: Activation Assay, Immunolabeling, Immunofluorescence, Labeling, Quantitation Assay

Journal: Neural Plasticity

Article Title: Activation of Sphingosine 1-Phosphate Receptor 1 Enhances Hippocampus Neurogenesis in a Rat Model of Traumatic Brain Injury: An Involvement of MEK/Erk Signaling Pathway

doi: 10.1155/2016/8072156

Figure Lengend Snippet: Activation of S1PR1 triggered MEK/Erk pathway phosphorylation in hippocampus at 7 days after TBI. (a) WB was performed to determine hippocampal S1PR1, pMEK, tMEK, pErk, and tErk expression in groups of sham, TBI+Vehicle, TBI+SEW, and TBI+SEW+VPC group ( n = 3 in sham and n = 6 in other groups). (b, c, and e) The level of S1PR1, pMEK, and pErk significantly increased after trauma. SEW2871 induced further upregulation of S1PR1, pMEK, and pErk. However, the effect was attenuated by administration of VPC23019. (d, f) Neither S1PR1 agonism nor antagonism had effect on tMEK and tErk expression in hippocampus after TBI. ∗ P < 0.05 versus sham group; # P < 0.05 versus TBI+Vehicle group or TBI+SEW+VPC group.

Article Snippet: Following blocked by 5% skim milk in Tris-buffered saline solution containing 0.1% Tween-20 (TBST), the membranes were incubated overnight at 4°C with the following primary antibodies: rabbit anti-rat S1PR1 antibody (1 : 500, Prosci, 4809, Poway, CA, USA), rabbit anti-rat phosphate Erk (pErk) antibody (1 : 1000, Cell Signaling, 9101, Beverly, MA, USA), rabbit anti-rat total Erk (tErk) antibody (1 : 1000, Cell Signaling, 9102, Beverly, MA, USA), rabbit anti-rat phosphate MEK (pMEK) antibody (1 : 1000, Cell Signaling, 9154, Beverly, MA, USA), and rabbit anti-rat total MEK (tMEK) antibody (1 : 1000, Cell Signaling, 9126, Beverly, MA, USA).

Techniques: Activation Assay, Phospho-proteomics, Expressing

Journal: Neural Plasticity

Article Title: Activation of Sphingosine 1-Phosphate Receptor 1 Enhances Hippocampus Neurogenesis in a Rat Model of Traumatic Brain Injury: An Involvement of MEK/Erk Signaling Pathway

doi: 10.1155/2016/8072156

Figure Lengend Snippet: S1PR1-induced NSCs proliferation and neuronal differentiation in TBI rats were affected by MEK/Erk activity. (a, b) Representative IF microphotographs of hippocampus immunolabeled with BrdU/SOX2 and BrdU/NeuN in TBI+SEW, TBI+SEW+U0126, TBI+VPC, and TBI+VPC+ERN groups at 7 days and 28 days after trauma ( n = 6 in each group). (c, d) Statistical analysis showed that, compared to TBI+SEW group, administration of U0126 significantly decreased the number of BrdU+/SOX2+ cells and BrdU+/NeuN+ cells in TBI+SEW+U0126 group. Reversely, the double-labeled cells in TBI+VPC+ERN group were significantly increased compared with TBI+VPC group. Scale bar: 50 μ m. ∗ P < 0.05 versus TBI+SEW group; # P < 0.05 versus TBI+VPC group.

Article Snippet: Following blocked by 5% skim milk in Tris-buffered saline solution containing 0.1% Tween-20 (TBST), the membranes were incubated overnight at 4°C with the following primary antibodies: rabbit anti-rat S1PR1 antibody (1 : 500, Prosci, 4809, Poway, CA, USA), rabbit anti-rat phosphate Erk (pErk) antibody (1 : 1000, Cell Signaling, 9101, Beverly, MA, USA), rabbit anti-rat total Erk (tErk) antibody (1 : 1000, Cell Signaling, 9102, Beverly, MA, USA), rabbit anti-rat phosphate MEK (pMEK) antibody (1 : 1000, Cell Signaling, 9154, Beverly, MA, USA), and rabbit anti-rat total MEK (tMEK) antibody (1 : 1000, Cell Signaling, 9126, Beverly, MA, USA).

Techniques: Activity Assay, Immunolabeling, Labeling

Journal: Neural Plasticity

Article Title: Activation of Sphingosine 1-Phosphate Receptor 1 Enhances Hippocampus Neurogenesis in a Rat Model of Traumatic Brain Injury: An Involvement of MEK/Erk Signaling Pathway

doi: 10.1155/2016/8072156

Figure Lengend Snippet: Cognitive function of rats in sham, TBI+Vehicle, TBI+SEW, TBI+SEW+U0126, TBI+VPC, and TBI+VPC+ERN group ( n = 3 in sham and n = 5 in other groups) were evaluated by Morris water maze (MWM) test. (a) Escape latency in hidden platform trial exhibited a gradual reduction tendency from 24 to 27 days after trauma. Daily escape latency of TBI+Vehicle group was longer than that of sham group. Use of S1PR1 agonist in TBI+SEW group significantly shorten the latency, but the effect was eliminated in TBI+SEW+U0126 group. In addition, the escape latency of TBI+VPC+ERN group decreased compared with TBI+VPC group. (b, c) Platform crossing times and target quadrant duration in probe trial revealed that, relative to sham group, TBI+Vehicle group presented less times and shorter duration at 28 days after TBI. S1PR1 activation significantly increased the two indexes of TBI+SEW group, but the favorable effect was blocked by U0126 treatment in TBI+SEW+U0126 group. Moreover, the times of platform crossing and duration in target quadrant of TBI+VPC group were lower than those of TBI+VPC+ERN group. (d) Rat swimming speed of the six groups did not show any statistical difference. ∗ P < 0.05 versus sham group; # P < 0.05 versus TBI+SEW group, † P < 0.05 versus TBI+VPC group.

Article Snippet: Following blocked by 5% skim milk in Tris-buffered saline solution containing 0.1% Tween-20 (TBST), the membranes were incubated overnight at 4°C with the following primary antibodies: rabbit anti-rat S1PR1 antibody (1 : 500, Prosci, 4809, Poway, CA, USA), rabbit anti-rat phosphate Erk (pErk) antibody (1 : 1000, Cell Signaling, 9101, Beverly, MA, USA), rabbit anti-rat total Erk (tErk) antibody (1 : 1000, Cell Signaling, 9102, Beverly, MA, USA), rabbit anti-rat phosphate MEK (pMEK) antibody (1 : 1000, Cell Signaling, 9154, Beverly, MA, USA), and rabbit anti-rat total MEK (tMEK) antibody (1 : 1000, Cell Signaling, 9126, Beverly, MA, USA).

Techniques: Activation Assay