Journal: Acta pharmaceutica Sinica. B

Article Title: S1PR1 serves as a viable drug target against pulmonary fibrosis by increasing the integrity of the endothelial barrier of the lung.

doi: 10.1016/j.apsb.2022.10.006

Figure Lengend Snippet: Figure 1 S1pr1 expressed in ECs of lung tissues and downregulated in IPF patients. The distribution of S1pr1 in human lung tissues (A, B). (A) Clusters of human lung tissues were identified as four cell types, including endothelial, epithelial, immune, and mesenchymal cells. (B) S1pr1 was highly expressed in endothelial (yellow) and immune cells (light green). The expression of S1pr1 in human lung tissues (C, D). (C) The RNA expression of S1pr1 in lung tissues between nonfibrotic individuals (n Z 10, Tobacco Users, 8 of 10) and IPF patients (n Z 12) were analyzed. (D) The RNA expression of S1pr1 in ECs between healthy individuals (n Z 10) and IPF patients (n Z 12) were measured. ****P < 0.0001 vs. control. (E) S1pr1 was highly expressed in lung ECs and immune cells (n Z 7). (F) Box and whisker plot showed the expression of S1pr1 in lung. (G) Real-time qPCR analysis of S1pr1 expression in primary fibroblasts, endothelial and epithelial cells of mouse lung tissues (n Z 3). Bars represent mean SEM, ****P < 0.0001 vs. fibroblasts.

Article Snippet: S1pr1 conditional genetically targeted (S1pr1þ/ ) mice and TekCreERT2 mice were generated by Cyagen Biosciences (Suzhou, China).

Techniques: Expressing, RNA Expression, Control, Whisker Assay

Journal: Acta pharmaceutica Sinica. B

Article Title: S1PR1 serves as a viable drug target against pulmonary fibrosis by increasing the integrity of the endothelial barrier of the lung.

doi: 10.1016/j.apsb.2022.10.006

Figure Lengend Snippet: Figure 2 S1pr1 deficiency in ECs worsened bleomycin-induced injury and fibrosis in mouse lungs. (A) S1pr1f/f mice were generated by gene targeting and they were crossed with the Tek-CreERT2 mice to obtain endothelial-specific S1pr1 knockout mice, which were named as S1pr1þ/

Article Snippet: S1pr1 conditional genetically targeted (S1pr1þ/ ) mice and TekCreERT2 mice were generated by Cyagen Biosciences (Suzhou, China).

Techniques: Generated, Knock-Out

Journal: Acta pharmaceutica Sinica. B

Article Title: S1PR1 serves as a viable drug target against pulmonary fibrosis by increasing the integrity of the endothelial barrier of the lung.

doi: 10.1016/j.apsb.2022.10.006

Figure Lengend Snippet: Figure 3 S1pr1 deficiency of ECs destroyed the integrity of vascular barrier and increased inflammation and fibrosis. (A) Heatmap of mRNA expression of CD31þ ECs of the lung (left) and volcano map of DEGs (middle). GO enrichment analysis (right) (n Z 3). (B) GSEA and heatmap of tight junction-related genes and heatmap of tight junction genes. (C) Western blot analysis of ZO-1 in the lung section (top). Statistical analysis of the expression of ZO-1 (left bottom). ZO-1 mRNA expression from transcriptome analysis (right bottom) (n Z 3). (D) Statistical analysis of CD45þ cell percentage of total cells (left) and MFI of a-SMA near CD31þ cells (right) (n Z 3). (E) Representative results for mIHC staining of CD45 in the lung sections from S1pr1þ/ con and Cre con mice. Nuclei were stained blue by DAPI, and the images were obtained at an original magnification of 20. (F) Representative results for mIHC staining of CD31 and a-SMA in the lung sections from S1pr1þ/ con and Cre con mice. Nuclei were stained blue by DAPI, and the images were obtained at an original magnification of 20. Bars represent mean SEM, *P < 0.05 and **P < 0.01 vs. Cre con group.

Article Snippet: S1pr1 conditional genetically targeted (S1pr1þ/ ) mice and TekCreERT2 mice were generated by Cyagen Biosciences (Suzhou, China).

Techniques: Expressing, Western Blot, Staining

Journal: Acta pharmaceutica Sinica. B

Article Title: S1PR1 serves as a viable drug target against pulmonary fibrosis by increasing the integrity of the endothelial barrier of the lung.

doi: 10.1016/j.apsb.2022.10.006

Figure Lengend Snippet: Figure 4 S1pr1 deficiency of ECs accelerated BLM-induced fibrosis in the lung. (A) Heatmap of mRNA expression of CD31þ ECs of the lung (left).VolcanomapofDEGs(middle).GOenrichmentanalysis(right)(B)GSEAwasperformed(nZ4).(C)Heatmapanalysisofcollagen(left),ECM (middle), and tight junction (right) related genes (n Z 4). (D) Representative results for mIHC staining of CD45þ in the lung sections from S1pr1þ/

Article Snippet: S1pr1 conditional genetically targeted (S1pr1þ/ ) mice and TekCreERT2 mice were generated by Cyagen Biosciences (Suzhou, China).

Techniques: Expressing, Staining

Journal: Acta pharmaceutica Sinica. B

Article Title: S1PR1 serves as a viable drug target against pulmonary fibrosis by increasing the integrity of the endothelial barrier of the lung.

doi: 10.1016/j.apsb.2022.10.006

Figure Lengend Snippet: Figure 5 Activation of S1PR1 by selective S1PR1 agonist, IMMH002, ameliorated mouse PF induced by BLM. (A) Survival curve of S1PR1 agonists on BLM-induced mouse fibrosis model. (B) Calculated lung index of animal model. Lung index Z weight of lung (g)/body weight (g) 1000 (n Z 7e10). (C) Lung function detected by pulmonary function test, including IC, Crs, Ras, Ers, Cst, G, H, and Est (n Z 3e5). (D) Histological analysis of the severity of lung fibrosis after BLM induction. Representative images for HE (top), Masson staining (bottom). Images were obtained at 200 magnification. (E) Statistical analysis of pathology score (left) and fibrosis score (right) (n Z 7e10). (F) Western blot analysis of ZO-1, E-cadherin, Snail, fibronectin, and b-actin (n Z 3). (G) Statistical analysis of Western blot. Bars represent mean SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. Model, #P < 0.05, ##P < 0.01 and ####P < 0.0001 vs. Sham.

Article Snippet: S1pr1 conditional genetically targeted (S1pr1þ/ ) mice and TekCreERT2 mice were generated by Cyagen Biosciences (Suzhou, China).

Techniques: Activation Assay, Animal Model, Staining, Western Blot

Journal: Acta pharmaceutica Sinica. B

Article Title: S1PR1 serves as a viable drug target against pulmonary fibrosis by increasing the integrity of the endothelial barrier of the lung.

doi: 10.1016/j.apsb.2022.10.006

Figure Lengend Snippet: Figure 6 The selective S1PR1 agonist, IMMH002, increase endothelial barrier. (A) Immunofluorescence staining of ZO-1 in HUVECs. Nuclei were stained blue by DAPI, and the images were obtained at an original magnification of 40 . (B) Statistical analysis of the MFI of ZO-1. (C) Relative EC permeability Z Fluorescence value of the lower chamber/Fluorescence value of the upper chamber and standardized by control groups. (D) TEER measurements were performed by the Millicell voltammeter. TEER (U$cm2) Z Resistance (U) 0.333 (R Z 3.25 mm, S Z 0.333 cm2) and standardized by control groups. Bars represent mean SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. PBS with thrombin; ###P < 0.001 and ####P < 0.0001 vs. Control. (E) KM mice were administrated with IMMH002 and DXM for 7 continuous days. Thereafter, mice were injected with 1% acetic acid to form a peritoneal leakage model. Peritoneal lavage fluid absorbance was measured at 590 nm (n Z 7e9). Bars represent mean SEM, *P < 0.05 vs. Control. (F) Visualization of endothelial cytoskeletal rearrangement. Nuclei were stained blue by DAPI, and the images were obtained at an original magnification of 600 . (G) Immunofluorescence staining of CD31 and ZO-1 in mouse lung sections. Nuclei were stained blue by DAPI, and the images were obtained at an original magnification of 600 .

Article Snippet: S1pr1 conditional genetically targeted (S1pr1þ/ ) mice and TekCreERT2 mice were generated by Cyagen Biosciences (Suzhou, China).

Techniques: Staining, Permeability, Fluorescence, Control, Injection

Journal: Acta pharmaceutica Sinica. B

Article Title: S1PR1 serves as a viable drug target against pulmonary fibrosis by increasing the integrity of the endothelial barrier of the lung.

doi: 10.1016/j.apsb.2022.10.006

Figure Lengend Snippet: Figure 7 An image illustrating how endothelial barrier was disrupted by S1pr1 deficiency. S1PR1 agonist, IMMH002, could ameliorate PF by enhancing endothelial tight junctions.

Article Snippet: S1pr1 conditional genetically targeted (S1pr1þ/ ) mice and TekCreERT2 mice were generated by Cyagen Biosciences (Suzhou, China).

Techniques:

Journal: Journal of Neuroinflammation

Article Title: Cry 2 deficiency leads to cognitive impairment through the microbiota-gut-brain axis mediated S1P/NLRP3/IL-1β pathway in mice

doi: 10.1186/s12974-026-03706-5

Figure Lengend Snippet: Gut microbiota induces sphingolipid pathway disturbance in the peripheral and brain. ( A ) Representative metabolites classified and counted based on their chemical taxonomy in shScr and shCry2 mice. The size of the pie chart corresponded to the relative abundance levels of metabolites, (n = 10 mice/group)., (B) Volcano plot of differentially expressed metabolites from shScr versus shCry2 mice. Blue, red, and grey represented downregulated, upregulated, and no significantly expressed metabolites, respectively, (n = 10 mice/group)., ( C ) Unsupervised hierarchical clustering of differentially expressed metabolites from shScr and shCry2 mice, (n = 10 mice/group)., ( D ) Pathway enrichment analysis of differentially expressed metabolites between shScr and shCry2 mice, (n = 10 mice/group)., ( E ) Representative Western blot and quantification of Sphk1 and S1PR1 in the hippocampus of shScr and shCry2 mice., ( F ) LC/MS analysis of Cer(18:1/18:0) and Cer(18:2/18:0) in shScr and shCry2 mice., ( G ) Linear regression analyses between sphingosine and Akkermansia, (left plot) and Ruminococcaceae, (right plot) expression levels from data integrated from 16 S rRNA sequencing and metabolomic sequencing. All data were expressed as mean ± SEM. Data were considered significant if * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Means were compared using the Student’s t-test in panel, ( E , F ). shScr: shScramble

Article Snippet: Then the membranes were incubated overnight at 4 °C with the primary antibodies [Tau5 (1:1000, ab80579, Abcam), Tau1 (1:1000, MAB3420, MilliporeSigma), pS396-tau (1:5000, ab109390, Abcam), pT231-tau (1:5000, ab151559, Abcam), pT181-tau (1:5000, ab254409, Abcam), pT217-tau (1:2000, 44–744, Invitrogen), Occludin (1:1000, ab216327, Abcam), ZO-1 (1:1000, ab307799, Abcam), Sphk1 (1:1000, 10670-1-AP, Proteintech), CRY2 (1:1000, 13997-1-AP, Proteintech), S1PR1 (1:1000, 55133-1-AP, Proteintech), β-actin (1:5000, 66009-1-Ig, Proteintech), NLRP3 (1:1000, ab263899, Abcam), IL-1β (1:1000, ab234437, Abcam)] diluted in 1×TBST and washed 10 min three times.

Techniques: Western Blot, Liquid Chromatography with Mass Spectroscopy, Expressing, Sequencing, Metabolomic, IF-P

Journal: Journal of Neuroinflammation

Article Title: Cry 2 deficiency leads to cognitive impairment through the microbiota-gut-brain axis mediated S1P/NLRP3/IL-1β pathway in mice

doi: 10.1186/s12974-026-03706-5

Figure Lengend Snippet: FTY720 ameliorated the cognitive decline, impairment of BBB and tau pathology. ( A ) An overview of the experimental design. After receiving stereotaxic brain injections, three-month-old mice were housed for six weeks, followed by intraperitoneal injections of saline or FTY720 for two weeks. Behavioral and molecular biology tests were then conducted., ( B - E ) Escape latency of the shScr-saline, shScr-FTY720, shCry2-saline, and shCry2-FTY720 mice for 5 consecutive days in the MWM test, ( B ). Percentage of time spent in the target quadrant in the spatial probe trail of the MWM test, ( C ). Number of platform crossings in the spatial probe trail of the MWM test, ( D ). Representative heatmaps of the swimming path in the spatial probe trail of day 6 in the MWM test, ( E ), (n = 11–12 mice/group)., ( F - H ) Representative Western blot and quantification of Occludin, ZO-1, ( F ), Sphk1, S1PR1, ( G ), Tau1, pS396-tau, Tau5, and pT231-tau, ( H ) in the hippocampus of shScr-saline, shScr-FTY720, shCry2-saline, and shCry2-FTY720 mice., ( I , J ) Representative immunofluorescence staining and quantification of pS396-tau, (red) in the hippocampus of shScr-saline, shScr-FTY720, shCry2-saline, and shCry2-FTY720 mice. Magnification × 10. Scale bar = 100 μm. All data were expressed as mean ± SEM. Data were considered significant if *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Means were compared using a repeated measures 2-way ANOVA test with Bonferroni post hoc comparison in panel, ( B ) and one-way ANOVA, ( C - D , F - I ). FI: Fluorescence intensity. shScr: shScramble

Article Snippet: Then the membranes were incubated overnight at 4 °C with the primary antibodies [Tau5 (1:1000, ab80579, Abcam), Tau1 (1:1000, MAB3420, MilliporeSigma), pS396-tau (1:5000, ab109390, Abcam), pT231-tau (1:5000, ab151559, Abcam), pT181-tau (1:5000, ab254409, Abcam), pT217-tau (1:2000, 44–744, Invitrogen), Occludin (1:1000, ab216327, Abcam), ZO-1 (1:1000, ab307799, Abcam), Sphk1 (1:1000, 10670-1-AP, Proteintech), CRY2 (1:1000, 13997-1-AP, Proteintech), S1PR1 (1:1000, 55133-1-AP, Proteintech), β-actin (1:5000, 66009-1-Ig, Proteintech), NLRP3 (1:1000, ab263899, Abcam), IL-1β (1:1000, ab234437, Abcam)] diluted in 1×TBST and washed 10 min three times.

Techniques: Saline, Western Blot, Immunofluorescence, Staining, IF-P, Comparison, Fluorescence

Journal: EMBO Reports

Article Title: S1P-S1PR1 signaling impairs CD8 + T cell metabolism and effector function in tumors

doi: 10.1038/s44319-026-00734-3

Figure Lengend Snippet: ( A – C ) Flow cytometric analysis of S1PR1 expression in tumor tissues and spleens of B6 mice bearing 15-day subcutaneously established tumors: ( A ) EL4 thymoma ( n = 4), (B) B16 melanoma ( n = 6), and ( C ) YUMM1.7 melanoma ( n = 7). ( D – F ) Assessment of S1PR1 expression in terminally exhausted (PD1⁺Tim3⁺) and progenitor-like exhausted (PD1⁻Tim3⁻) CD8⁺ T cells isolated from ( D ) EL4 thymoma ( n = 4, P = 0.0005), ( E ) B16 melanoma ( n = 6), and ( F ) YUMM1.7 melanoma ( n = 7). ( G ) Purified CD8⁺ T cells from wild-type B6 mice were activated in vitro for 3 days, and transcript levels of S1pr1-S1pr5 were quantified by qPCR ( n = 4). ( H ) Purified CD8⁺ T cells from wild-type B6 mice were activated in vitro for 3 days, and cell-surface S1PR1 expression was examined by flow cytometry ( n = 3). ( I ) Flow cytometry-based intracellular cytokine analysis of in vitro-activated murine CD8⁺ T cells ( n = 7). ( J ) Proliferative capacity of CTV-labeled mouse CD8⁺ T cells activated in the presence or absence of S1P, assessed using flow cytometry ( n = 3). ( K ) Flow cytometry analysis of apoptosis in mouse CD8⁺ T cells activated with or without S1P, quantified by Annexin V and 7-AAD staining ( n = 5). ( L – N ) Purified mouse CD8⁺ T cells activated in the presence or absence of S1P were subjected to metabolic flux analysis to determine: ( L ) extracellular acidification rate (ECAR) in response to glucose, oligomycin, and 2-deoxyglucose (2DG); ( M ) basal glycolytic rate following glucose addition ( n = 4); and ( N ) glycolytic capacity ( n = 4). ( O ) Heatmap illustrating transcript levels of key glycolysis-associated genes in S1P-treated versus vehicle-treated mouse CD8⁺ T cells ( n = 4). ( P – S ) Oxygen consumption rate (OCR) of mouse CD8⁺ T cells activated in the presence or absence of S1P under basal conditions and after sequential addition of mitochondrial inhibitors ( P ), along with quantification of ( Q ) basal respiration ( n = 4), ( R ) maximal respiratory capacity ( n = 4), and ( S ) spare respiratory capacity ( n = 4, P = 0.0021). * P < 0.05; ** P < 0.01; *** P < 0.005; **** P < 0.0001; ns, nonsignificant ( P > 0.05), the error bar represents the standard deviation (SD). P values are derived from unpaired two-tailed Student’s t test ( A – F , I , K , M , N , Q – S ), one-way ANOVA ( H ), and two-way ANOVA test ( G , J , O ). .

Article Snippet: Cells were maintained in complete DMEM (Gibco, Thermo Fisher Scientific) supplemented with:10% fetal bovine serum (FBS; Gibco), 1% penicillin–streptomycin (Gibco) For lentiviral production, HEK293T cells were co-transfected with: 15 μg lentiviral expression plasmid encoding S1pr1 shRNA, Eif2ak3 shRNA, or non-targeting scrambled shRNA control (Origene, USA) 10 μg psPAX2 packaging plasmid 5 μg pMD2.G envelope plasmid Transfection was carried out using the CaCl2/HBS precipitation method.

Techniques: Expressing, Isolation, Purification, In Vitro, Flow Cytometry, Labeling, Staining, Standard Deviation, Derivative Assay, Two Tailed Test

Journal: EMBO Reports

Article Title: S1P-S1PR1 signaling impairs CD8 + T cell metabolism and effector function in tumors

doi: 10.1038/s44319-026-00734-3

Figure Lengend Snippet: ( A – C ) Gating strategy used for flow cytometry analysis of heterogeneous exhaustion states of CD8⁺ T cells within the tumor microenvironment (TME) of (A) EL4 thymoma ( n = 4), ( B ) B16F10 melanoma ( n = 5), ( C ) YUMM1.7 melanoma ( n = 7), distinguished by the differential expression of PD1 and Tim3. ( D – H ) Human CD8⁺ T cells isolated from healthy donor PBMCs were activated for 3 days and then subjected to either continuous TCR stimulation or cultured (without TCR stimulation) with IL-2 for 15 days. Cells were analyzed for ( D ) exhaustion-associated surface markers, ( E ) intracellular production of IFNγ and TNFα, ( F ) intracellular expression of transcription factor TCF1, ( G ) intracellular expression of cell proliferation marker Ki67, and ( H ) cell death using Annexin V and 7AAD. The adjacent bar diagram represents cumulative data from n = 4 biological replicates. ( I – M ) CD8⁺ T cells isolated from the spleen of wild-type B6 mice were activated for 2 days and then subjected to either continuous TCR stimulation or cultured (without TCR stimulation) with IL-2 culture for 9 days. Cells were assessed for ( I ) exhaustion-associated surface markers, ( J ) intracellular production of IFNγ and TNFα, ( K ) intracellular expression of the transcription factor TCF1, ( L ) intracellular expression of cell proliferation marker Ki67, and ( M ) cell death using Annexin V and 7AAD. The adjacent bar diagram represents cumulative data from n = 4 biological replicates. ( N , O ) Flow cytometry analysis of S1PR1 expression in acutely versus chronically stimulated human ( N ) and murine ( O ) CD8⁺ T cells. The adjacent bar diagram represents cumulative data from n = 4 biological replicates. ( P ) Flow cytometry analysis of p-STAT3 expression in CD8⁺ T cells isolated from spleens versus tumor tissues, with adjacent bar graphs representing cumulative results from four biological replicates ( n = 4). ( Q ) Purified mouse CD8⁺ T cells activated in the presence or absence of S1P were assessed for flow cytometry-based expression of CD25. The adjacent bar diagram represents cumulative data from three biological replicates ( n = 3). * P < 0.05; ** P < 0.01; *** P < 0.005; **** P < 0.0001; ns, nonsignificant ( P > 0.05), the error bar represents the standard deviation (SD). P values are derived from unpaired two-tailed Student’s t test ( A – P ). .

Article Snippet: Cells were maintained in complete DMEM (Gibco, Thermo Fisher Scientific) supplemented with:10% fetal bovine serum (FBS; Gibco), 1% penicillin–streptomycin (Gibco) For lentiviral production, HEK293T cells were co-transfected with: 15 μg lentiviral expression plasmid encoding S1pr1 shRNA, Eif2ak3 shRNA, or non-targeting scrambled shRNA control (Origene, USA) 10 μg psPAX2 packaging plasmid 5 μg pMD2.G envelope plasmid Transfection was carried out using the CaCl2/HBS precipitation method.

Techniques: Flow Cytometry, Quantitative Proteomics, Isolation, Cell Culture, Expressing, Marker, Purification, Standard Deviation, Derivative Assay, Two Tailed Test

Journal: EMBO Reports

Article Title: S1P-S1PR1 signaling impairs CD8 + T cell metabolism and effector function in tumors

doi: 10.1038/s44319-026-00734-3

Figure Lengend Snippet: ( A ) Western blot analysis showing expression of SphK1 in wild-type and SphK1 KO YUMM1.7 cells ( n = 3). ( B ) Flow cytometry analysis of the surface expression of S1PR1 on intratumoral CD8 + T cells following administration of S1PR1 antagonist W146 or vehicle control in YUMM1.7 tumor-bearing mice ( n = 4). * P < 0.05; ** P < 0.01; *** P < 0.005; **** P < 0.0001; ns, nonsignificant ( P > 0.05), the error bar represents the standard deviation (SD). P values are derived from unpaired two-tailed Student’s t test ( B ). .

Article Snippet: Cells were maintained in complete DMEM (Gibco, Thermo Fisher Scientific) supplemented with:10% fetal bovine serum (FBS; Gibco), 1% penicillin–streptomycin (Gibco) For lentiviral production, HEK293T cells were co-transfected with: 15 μg lentiviral expression plasmid encoding S1pr1 shRNA, Eif2ak3 shRNA, or non-targeting scrambled shRNA control (Origene, USA) 10 μg psPAX2 packaging plasmid 5 μg pMD2.G envelope plasmid Transfection was carried out using the CaCl2/HBS precipitation method.

Techniques: Western Blot, Expressing, Flow Cytometry, Control, Standard Deviation, Derivative Assay, Two Tailed Test

Journal: EMBO Reports

Article Title: S1P-S1PR1 signaling impairs CD8 + T cell metabolism and effector function in tumors

doi: 10.1038/s44319-026-00734-3

Figure Lengend Snippet: ( A ) Western blot analysis showing expression of CHOP in control, S1P, and GSK pretreated S1P-treated CD8 + T cells. The adjacent bar graph depicts normalized densitometric data from three biological replicates ( n = 3). ( B ) q-PCR analysis of Ddit3 (encoding CHOP) in respective groups ( n = 3). ( C , D ) Western blot analysis showing expression of CHOP in activated CD8 + T cells upon S1pr1 knockdown using ( C ) siRNA and ( D ) shRNA. The adjacent bar graph depicts normalized densitometric data from three biological replicates ( N = 3, for both ( C , D ). ( E ) Purified mouse CD8⁺ T cells activated under the indicated treatment conditions were analyzed for the production of effector cytokines. The adjacent bar plots represent cumulative data from three biological replicates ( n = 3). ( F ) Purified mouse CD8⁺ T cells activated under the indicated treatment conditions were assessed for the frequency of CD8 + T cells undergoing apoptosis, as determined by Annexin V and 7AAD staining. The adjacent bar plots represent cumulative data from four biological replicates ( n = 4). ( G – L ) C57BL/6 mice ( n = 4 mice/group) with subcutaneously established YUMM1.7 melanoma tumor treated either with vehicle control or GSK, as ( G ) represented schematically, were evaluated for: ( H ) tumor growth, ( I ) the ability of CD8 + T cells from the tumor site to produce different effector cytokines, ( J ) frequency of CD8 + T cells at the tumor site, ( K ) expression of PD1, and ( L ) expression of Tim3 on intratumoral CD8 + T cells. * P < 0.05; ** P < 0.01; *** P < 0.005; **** P < 0.0001; ns, nonsignificant ( P > 0.05), the error bar represents the standard deviation (SD). P values are derived from unpaired two-tailed Student’s t test ( I – L ), one-way ANOVA ( A – F ), and two-way ANOVA test ( H ). .

Article Snippet: Cells were maintained in complete DMEM (Gibco, Thermo Fisher Scientific) supplemented with:10% fetal bovine serum (FBS; Gibco), 1% penicillin–streptomycin (Gibco) For lentiviral production, HEK293T cells were co-transfected with: 15 μg lentiviral expression plasmid encoding S1pr1 shRNA, Eif2ak3 shRNA, or non-targeting scrambled shRNA control (Origene, USA) 10 μg psPAX2 packaging plasmid 5 μg pMD2.G envelope plasmid Transfection was carried out using the CaCl2/HBS precipitation method.

Techniques: Western Blot, Expressing, Control, Knockdown, shRNA, Purification, Staining, Standard Deviation, Derivative Assay, Two Tailed Test

Journal: EMBO Reports

Article Title: S1P-S1PR1 signaling impairs CD8 + T cell metabolism and effector function in tumors

doi: 10.1038/s44319-026-00734-3

Figure Lengend Snippet: ( A ) Western blot analysis of phospho-p38 (p-p38) and total p38 expression, in vehicle control and S1P-treated CD8 + T cells. The adjacent bar graph depicts normalized densitometric data from three biological replicates ( n = 3). ( B , C ) Western blot analysis showing the expression of p-p38 and total p38 in activated T cells upon S1pr1 knockdown using ( B ) siRNA ( n = 3) and (C) shRNA ( n = 3). The adjacent bar graph depicts normalized densitometric data. ( D ) Western blot analysis of p-p38 and total p38 in CD8 + T cells activated in the presence or absence of S1P, along with the indicated inhibitor. The adjacent bar graph depicts normalized densitometric data from three biological replicates ( n = 3). ( E , F ) qPCR analysis of transcript levels of different ( E ) Map3k and (F) Map2k genes in CD8 + T cells in respective groups ( n = 4). ( G ) CD8 + T cells were activated in the presence or absence of S1P and were collected and processed for chromatin-immunoprecipitation (ChIP) assay with an antibody specific for CHOP or with rabbit IgG control. qPCR primers specific for the known CHOP binding gene ( Dr5 ) and different Map3K and Map2K , along with Mapk14 , were used to determine CHOP binding to the respective promoters ( n = 4). ( H , I ) Purified mouse CD8⁺ T cells activated under the indicated treatment conditions were assessed for: ( H ) T cell death by Annexin V and 7AAD staining and ( I ) frequency of CD8 + T cells producing different effector cytokines. The adjacent bar represents cumulative data from four biological replicates ( n = 4, for both ( H , I )). ( J ) Extracellular flux assay for determining of oxygen consumption rate (OCR) in activated CD8 + T cells in respective groups. * P < 0.05; ** P < 0.01; *** P < 0.005; **** P < 0.0001; ns, nonsignificant ( P > 0.05), the error bar represents the standard deviation (SD). P values are derived from unpaired two-tailed Student’s t test ( A ), one-way ANOVA ( B – D , H , I ), and two-way ANOVA test ( E – G ). .

Article Snippet: Cells were maintained in complete DMEM (Gibco, Thermo Fisher Scientific) supplemented with:10% fetal bovine serum (FBS; Gibco), 1% penicillin–streptomycin (Gibco) For lentiviral production, HEK293T cells were co-transfected with: 15 μg lentiviral expression plasmid encoding S1pr1 shRNA, Eif2ak3 shRNA, or non-targeting scrambled shRNA control (Origene, USA) 10 μg psPAX2 packaging plasmid 5 μg pMD2.G envelope plasmid Transfection was carried out using the CaCl2/HBS precipitation method.

Techniques: Western Blot, Expressing, Control, Knockdown, shRNA, Chromatin Immunoprecipitation, Binding Assay, Purification, Staining, XF Assay, Standard Deviation, Derivative Assay, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: Immunomodulation Eliminates Inflammation in the Hippocampus in Experimental Autoimmune Encephalomyelitis, but Does Not Ameliorate Anxiety-Like Behavior

doi: 10.3389/fimmu.2021.639650

Figure Lengend Snippet: Target genes and their primer pairs.

Article Snippet: Immunostaining of hippocampal sections was performed as described ( , ) using polyclonal antibodies to CD3 (at 1:400, Dako, Glostrup, Denmark), ionized calcium-binding adapter molecule 1 ([Iba1] at 1:200, Wako Chemical Industries, Japan), and monoclonal antibodies against MAP2 (at 1:500 Novus Biologicals, Centennial, CO), S1PR1 ([EDG1] at 1:100, OriGene Technologies Inc., Rockford, MD), S1PR3 and S1PR5 ([EDG3 and EDG8], TNF-α and IFN-γ all at 1:100, Novus Biologicals, Abingdon, UK).

Techniques: Sequencing

Journal: Frontiers in Immunology

Article Title: Immunomodulation Eliminates Inflammation in the Hippocampus in Experimental Autoimmune Encephalomyelitis, but Does Not Ameliorate Anxiety-Like Behavior

doi: 10.3389/fimmu.2021.639650

Figure Lengend Snippet: EAE effect on S1PR1, S1PR3 and S1PR5 expression in the hippocampus. Comparison of S1PR expression between sham (purple) and EAE (orange) groups. The left-hand panel shows the regions of interest (Aa-c, Ba-c, Ca-c) and the direction of change in three hippocampal sub-fields for each S1PR (downregulation = dotted arrow, upregulation = dashed arrow, or no change = horizontal arrow). The main panel shows immunostained sections with anti-GFAP (red) and anti-S1PR1 (green; Ac-e, f-h ), anti-S1PR3 (green; Bc-e, f-h ) or anti-S1PR5 (green; Cc-e, f-h ); nuclei were stained with DAPI. No primary antibody controls are shown in Ai-j, Bi-j and Ci-j ). Scale bars = 10 µm. Experiments were replicated 3 times. Quantification of images is shown in Figure 7 .

Article Snippet: Immunostaining of hippocampal sections was performed as described ( , ) using polyclonal antibodies to CD3 (at 1:400, Dako, Glostrup, Denmark), ionized calcium-binding adapter molecule 1 ([Iba1] at 1:200, Wako Chemical Industries, Japan), and monoclonal antibodies against MAP2 (at 1:500 Novus Biologicals, Centennial, CO), S1PR1 ([EDG1] at 1:100, OriGene Technologies Inc., Rockford, MD), S1PR3 and S1PR5 ([EDG3 and EDG8], TNF-α and IFN-γ all at 1:100, Novus Biologicals, Abingdon, UK).

Techniques: Expressing, Comparison, Staining

Journal: Frontiers in Immunology

Article Title: Immunomodulation Eliminates Inflammation in the Hippocampus in Experimental Autoimmune Encephalomyelitis, but Does Not Ameliorate Anxiety-Like Behavior

doi: 10.3389/fimmu.2021.639650

Figure Lengend Snippet: Quantification of S1PR1, S1PR3 and S1PR5 expression in the hippocampus in EAE and following FTY720 treatment. (A) shows the EAE effect (EAE vs sham, images from Figure 4 ) from each hippocampal sub-region, with sham (purple) and EAE (orange), with signals captured using an arbitrary scale and estimated by ImageJ. (B) shows the direct FTY720 effect on sham mice (sham vs sham+FTY720, images from Figure 5 ) on S1PR1, S1PR3 and S1PR5 expression in the hippocampus with sham (purple), sham+FTY720-L (light blue) and sham+FTY720-H (dark blue). (C) shows the combined EAE and FTY720 effects (EAE vs EAE+FTY720, images from Figure 6 ) on S1PR1, S1PR3 and S1PR5 expression in the hippocampus with EAE (orange), EAE+FTY720-L (light grey) and EAE+FTY720-H (dark grey). N = 4 mice/group × 6 sections/mouse (*p<0.05). Experiments were replicated 3 times. Data were analyzed using one-way ANOVA and shown as mean ± standard error of the mean (SEM).

Article Snippet: Immunostaining of hippocampal sections was performed as described ( , ) using polyclonal antibodies to CD3 (at 1:400, Dako, Glostrup, Denmark), ionized calcium-binding adapter molecule 1 ([Iba1] at 1:200, Wako Chemical Industries, Japan), and monoclonal antibodies against MAP2 (at 1:500 Novus Biologicals, Centennial, CO), S1PR1 ([EDG1] at 1:100, OriGene Technologies Inc., Rockford, MD), S1PR3 and S1PR5 ([EDG3 and EDG8], TNF-α and IFN-γ all at 1:100, Novus Biologicals, Abingdon, UK).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Immunomodulation Eliminates Inflammation in the Hippocampus in Experimental Autoimmune Encephalomyelitis, but Does Not Ameliorate Anxiety-Like Behavior

doi: 10.3389/fimmu.2021.639650

Figure Lengend Snippet: Direct FTY720 effect on S1PR1, S1PR3 and S1PR5 expression in the hippocampus. Comparison of S1PR expression between sham, sham+FTY720-Lo and sham+FTY720-H groups. The left-hand panel shows the regions of interest (Aa-c, Ba-c, Ca-c) and the direction of change in three hippocampal sub-fields for each S1PR (downregulation = dotted arrow, upregulation = dashed arrow, or no change = horizontal arrow). The main panel shows immunostained sections with anti-GFAP (red) and anti-S1PR1 (green; Ad-f, g-i ), anti-S1PR3 (green; Bd-f, g-i ) or anti-S1PR5 (green; Cd-f, g-i) ; nuclei were stained with DAPI. Original images for Aa, Ba and Ca are found in Figure 4 . No primary antibody controls are shown in Aj-k, Bj-k and Cj-k ). N = 4 mice/group × 6 sections/mouse. Scale bars = 10 µm. Experiments were replicated 3 times. Quantification of images is shown in Figure 7 .

Article Snippet: Immunostaining of hippocampal sections was performed as described ( , ) using polyclonal antibodies to CD3 (at 1:400, Dako, Glostrup, Denmark), ionized calcium-binding adapter molecule 1 ([Iba1] at 1:200, Wako Chemical Industries, Japan), and monoclonal antibodies against MAP2 (at 1:500 Novus Biologicals, Centennial, CO), S1PR1 ([EDG1] at 1:100, OriGene Technologies Inc., Rockford, MD), S1PR3 and S1PR5 ([EDG3 and EDG8], TNF-α and IFN-γ all at 1:100, Novus Biologicals, Abingdon, UK).

Techniques: Expressing, Comparison, Staining

Journal: Frontiers in Immunology

Article Title: Immunomodulation Eliminates Inflammation in the Hippocampus in Experimental Autoimmune Encephalomyelitis, but Does Not Ameliorate Anxiety-Like Behavior

doi: 10.3389/fimmu.2021.639650

Figure Lengend Snippet: Combined EAE and FTY720 effects on S1PR1, S1PR3 and S1PR5 expression in the hippocampus. Comparison of S1PR expression between EAE, EAE+FTY720-L and EAE+FTY720-H groups. The left-hand panel shows the regions of interest (Aa-c, Ba-c, Ca-c) and the direction of change in three hippocampal sub-fields for each S1PR (downregulation = dotted arrow, upregulation = dashed arrow, or no change = horizontal arrow). The main panel shows immunostained sections with anti-GFAP (red) and anti-S1PR1 (green; Ad-f, g-i ), anti-S1PR3 (green; Bd-f, g-i ) or anti-S1PR5 (green; Cd-f, g-i) ; nuclei were stained with DAPI. Original images for Aa, Ba and Ca are found in Figure 4 . No primary antibody controls are shown in Aj-k, Bj-k and Cj-k ). N = 4 mice/group × 6 sections/mouse. Scale bars = 10 µm. Experiments were replicated 3 times. Quantification of images is shown in Figure 7 .

Article Snippet: Immunostaining of hippocampal sections was performed as described ( , ) using polyclonal antibodies to CD3 (at 1:400, Dako, Glostrup, Denmark), ionized calcium-binding adapter molecule 1 ([Iba1] at 1:200, Wako Chemical Industries, Japan), and monoclonal antibodies against MAP2 (at 1:500 Novus Biologicals, Centennial, CO), S1PR1 ([EDG1] at 1:100, OriGene Technologies Inc., Rockford, MD), S1PR3 and S1PR5 ([EDG3 and EDG8], TNF-α and IFN-γ all at 1:100, Novus Biologicals, Abingdon, UK).

Techniques: Expressing, Comparison, Staining