Journal: bioRxiv

Article Title: Tension sensing by FAK governs nuclear mechanotransduction, endothelial transcriptome and fate

doi: 10.1101/2023.04.24.538195

Figure Lengend Snippet: Protocol of inhibiting DNMT3a using TF-3 (1 mg/kg , i.v ) in WT mice. After 1 h of TF-3 administration the mice received LPS (10 mg/kg, i.p ) and lungs were harvested after 8 h (n=5mice/group). DNMT activity (B) , mRNA expression of KLF2 (C) and lung edema (D) were determined (n=3). E, mRNA expression of indicated genes in control, FAK depleted, and FAK + DNMT3a-depleted EC. GAPDH was used as an internal control (n=3). F, Schematics showing generation of EC-Fak/Dnmt3a -/- (double knockout) mice. Offspring of Fak fl/fl and Dnmt3a fl/fl mice were bred with SCL-Cre ERT to generate Fak/Dnmt3a fl SCL CreERT mice. Tamoxifen (80 mg/kg, i.p) was injected into 4-week old Fak/Dnmt3a fl SCL- CreERT mice for five consecutive days followed by 7-day drug washout period. G, mRNA expression shows deletion of FAK and S1PR1 in lungs from double knockout versus control mice. Experiments were repeated three times independently. H, Lung edema was assessed by measuring lung weight-dry weight ratio in Fak fl/fl , EC-Fak -/- and EC-FAK-null mice receiving TF-3 (1 mg/kg, i.v). I, mRNA expression of indicated genes in lungs of Fak fl/fl , EC-Fak -/- and EC- Fak -/- /Dnmt3a -/- . GAPDH was used as an internal control (n=3). J-K, TEER assay in FAK- depleted EC without or with TF-3 (1 µM) treatment as described in J . Data in plots B, C, D, E, G, H, I and K show mean ± SEM. Statistical significance was assessed by One-way ANOVA followed by post hoc Tukey’s test for B, C, D, E, H, I and K, while unpaired t test was used for G . **** P < 0.0001 relative to siCtr (K), *** P < 0.001 relative to no treatment (C), Fak fl/fl (G, H), ** P < 0.01 to no treatment (B, D), siCtr (E) fl/fl (I), * P < 0.05 to siCtr (E), fl/fl (I). # P < 0.05 to LPS treated (B, D), ## P < 0.01 to no treatment (C) and ### P < 0.001 to EC-Fak -/- . Also see Supplementary Fig. 4.

Article Snippet: Anti-S1PR1 antibody (Cat #ASR-011) was acquired from Alomone Labs. Anti-Emerin (Cat #30853), anti-Src (Cat #2109), p-Src (Cat #12432), anti-Fak (Cat #3285T), anti-Lamin (Cat #86846S), anti- DNMT3a (Cat # 3598) antibodies from Cell Signaling Technology whereas, anti-actin (Cat #sc- 47778), anti-DNMT3b (Cat #sc-20704), anti-p-Tyr (PY99, Cat #sc-7020) and anti-p-Tyr (PY20, Cat # sc-508) were acquired from Santa Cruz Biotechnology.

Techniques: Activity Assay, Expressing, Control, Double Knockout, Injection

Journal: bioRxiv

Article Title: Tension sensing by FAK governs nuclear mechanotransduction, endothelial transcriptome and fate

doi: 10.1101/2023.04.24.538195

Figure Lengend Snippet: Volcano plot of differential genes from bulk RNA- seq analysis of EC sorted from Fak fl/fl and EC-Fak -/- mice lungs. S1PR1 mRNA (B) or protein (C) expression in FAK + versus FAK - EC. GAPDH was used an internal control for mRNA while actin was used as a loading control (n=3). D, Corresponding densitometry of the blots. E, S1P (1 mg/kg, i.v ) was administered to Fak fl/fl or EC-Fak -/- mice and after 30 min lung edema was assessed by measuring wet-to-dry weight ratio of the lungs (n=5 mice/group). F, A representative micrograph shows effect of S1P in re-assembling adherens junctions in control versus FAK-depleted EC. EC received 1 µM S1P for 15 min after which the cells were co-immunostained with anti-VE-cadherin and anti-FAK antibodies. Images were acquired using confocal microscope. Note, Control EC were plated at sub-confluent level to match gaps in FAK depleted EC before S1P stimulation (n=3). Scale bar 20 µm. G, Plot shows intercellular gap in control and FAK-depleted EC after with or without 1 µM S1P for 15 min. Gap area was assessed by ImageJ. The experiment was independently repeated three times. H, TEER in control and FAK-depleted EC at baseline and after addition of 1 µM S1P ( n = 8 wells/group). The assay was repeated three times independently. I-J, EC plated on TEER electrodes were processed as in H for 48 h. EC were then re-transfected with vector or GFP-tagged WT-S1PR1 cDNA and TEER was measured (I) and S1PR1 expression (J) was quantified after 72 h after addition of 1 µM S1P ( n = 8wells/group). The assay was repeated three times independently. Immunoblot of EC shows S1PR1 expression taking actin as a loading control (n=3). K, Edema measurement was performed in Fak fl/fl and EC-Fak -/- mice 48 h after delivery of liposomes containing VE-cadherin promoter driven GFP-tagged WT-S1PR1 cDNA as in 1K (n=6 mice/group). Data in plots B , D , E , G and K show mean ± SEM. Statistical significance was assessed by one-way ANOVA followed by Post hoc Tukey’s test in E and K, while unpaired t-test was used in B, D and G. ** P < 0.01 relative to FAK fl/fl (B, D) and Fak fl/fl and EC-Fak -/- receiving empty vector (K) * P < 0.05 relative to siCtr (G). Also see Supplementary Fig. 5.

Article Snippet: Anti-S1PR1 antibody (Cat #ASR-011) was acquired from Alomone Labs. Anti-Emerin (Cat #30853), anti-Src (Cat #2109), p-Src (Cat #12432), anti-Fak (Cat #3285T), anti-Lamin (Cat #86846S), anti- DNMT3a (Cat # 3598) antibodies from Cell Signaling Technology whereas, anti-actin (Cat #sc- 47778), anti-DNMT3b (Cat #sc-20704), anti-p-Tyr (PY99, Cat #sc-7020) and anti-p-Tyr (PY20, Cat # sc-508) were acquired from Santa Cruz Biotechnology.

Techniques: RNA Sequencing, Expressing, Control, Microscopy, Transfection, Plasmid Preparation, Western Blot, Liposomes

Journal: bioRxiv

Article Title: Tension sensing by FAK governs nuclear mechanotransduction, endothelial transcriptome and fate

doi: 10.1101/2023.04.24.538195

Figure Lengend Snippet: A, Schematic representation of S1PR1 promoter with three KLF2 binding sites (-341, -501, and -937 from TSS). B, EC were co-transfected with WT-S1PR1 luciferase promoter or mutated S1PR1 luciferase promoter (which lacks three KLF2 binding sites) along with KLF2 cDNA (n=3). Luciferase activity was determined after 24 h. C-D, EC were transfected with control shRNA or KLF2 shRNA. After 48 h, S1PR1 mRNA (C) or protein expression (D) was determined. GAPDH was used as a loading control for mRNA and actin was used as loading control for protein (n=3). E, Corresponding densitometry of the above blots. F, EC plated on TEER electrodes were transfected with control or KLF2 shRNA. After 48 h, cells were stimulated with S1P (1 µM) and TEER was measured as in 2G (n = 8wells/group). G, After 48 h of siFAK transfection, EC were co-transfected with vector or KLF2 cDNA. mRNA expression of indicated genes was assessed by qPCR. GAPDH was used an internal control (n=3). H-I, EC plated on TEER electrodes were transfected with control or FAK siRNA. After 48 h, cells were co-transfected with WT-KLF2 cDNA. After 72 h cells were stimulated with S1P, and TEER was assessed as in ( n = 8wells/group). J-K, S1PR1 mRNA expression in FAK-depleted EC after DNMT3a knockdown (J) or inhibition with 1 µM theaflavin-3,3’-digallate (TF-3) for 4 h. (K). L , S1PR1 mRNA in EC-Fak -/- mice after 24 h injection of AZA (0.5 mg/kg, i.v. ) or TF-3 (1mg/kg, i.v ). GAPDH was used an internal control (n=3). Data plotted in B, C, E, G, I, J, K and L are given as mean ± SEM. Statistical significance was assessed by one-way ANOVA followed by Post hoc Tukey’ test in B, G, I, J, K and L and unpaired t test was used in C and E. **** P < 0.0001 relative to siFAK+KLF2 (I), *** P < 0.001 to WT-S1PR1 (B), shCtr (C) Fak fl/fl (L), ** P < 0.01 relative to shCtr (E), siCtr (G, J) siCtr+TF-3 (K) and * P < 0.05 siCtr (G), siCtr+TF-3 (K). Also see Supplementary Fig. 5 .

Article Snippet: Anti-S1PR1 antibody (Cat #ASR-011) was acquired from Alomone Labs. Anti-Emerin (Cat #30853), anti-Src (Cat #2109), p-Src (Cat #12432), anti-Fak (Cat #3285T), anti-Lamin (Cat #86846S), anti- DNMT3a (Cat # 3598) antibodies from Cell Signaling Technology whereas, anti-actin (Cat #sc- 47778), anti-DNMT3b (Cat #sc-20704), anti-p-Tyr (PY99, Cat #sc-7020) and anti-p-Tyr (PY20, Cat # sc-508) were acquired from Santa Cruz Biotechnology.

Techniques: Binding Assay, Transfection, Luciferase, Activity Assay, Control, shRNA, Expressing, Plasmid Preparation, Knockdown, Inhibition, Injection

Journal: bioRxiv

Article Title: Tension sensing by FAK governs nuclear mechanotransduction, endothelial transcriptome and fate

doi: 10.1101/2023.04.24.538195

Figure Lengend Snippet: DNMT activity was measured in FAK depleted EC transduced with WT-emerin or phosphodefective emerin mutant after 24 h post-transfection as in (n=3). B, mRNA expression of KLF2 and S1PR1 in FAK depleted EC transducing WT-or phosphodefective-emerin. GAPDH was used as an internal control (n=3). C, Representative micrograph demonstrates intercellular gap as measured by immunostaining with anti-VE-Cadherin antibody in FAK-depleted EC after transduction with WT- or phosphodefective-emerin mutant. Scale bar 20 µm. D, Intercellular gap was measured as in 4F. E, EC plated on TEER electrodes were transfected with either Ctr or FAK siRNA and after 48h WT- or phosphodefective- emerin mutant was transduced. TEER was assessed after 72 h ( n = 8/group). The assay was repeated three times independently. Data plotted in A, B, D and E are given as mean ± SEM. Statistical significance was assessed by one-way ANOVA followed by Post hoc Tukey’s test. **** P < 0.0001 relative to siCtr (E), *** P < 0.001 siCtr (A, B), ** P < 0.01 siCtr (D), * P < 0.05 siCtr (D). F, Model of FAK suppression of nuclear mechanotransduction and synthesis of genes maintaining vascular barrier. We posit that loss of FAK increases RhoA activity which in turn increases intracellular mechanical tension by activating Rho kinase and myosin ATPase activity. Increased tension activates cSrc which phosphorylates emerin. Phosphorylated emerin activates DNMT3a, which in turn methylates the KLF2 promoter, blocking binding of its transcription factor, MEF2, and hence KLF2 synthesis. Suppression of KLF2 synthesis compromises S1PR1 transcription leading to conversion of restrictive EC to leaky EC which impairs vascular homeostasis.

Article Snippet: Anti-S1PR1 antibody (Cat #ASR-011) was acquired from Alomone Labs. Anti-Emerin (Cat #30853), anti-Src (Cat #2109), p-Src (Cat #12432), anti-Fak (Cat #3285T), anti-Lamin (Cat #86846S), anti- DNMT3a (Cat # 3598) antibodies from Cell Signaling Technology whereas, anti-actin (Cat #sc- 47778), anti-DNMT3b (Cat #sc-20704), anti-p-Tyr (PY99, Cat #sc-7020) and anti-p-Tyr (PY20, Cat # sc-508) were acquired from Santa Cruz Biotechnology.

Techniques: Activity Assay, Transduction, Mutagenesis, Transfection, Expressing, Control, Immunostaining, Blocking Assay, Binding 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 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: 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

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: Journal of Experimental & Clinical Cancer Research : CR

Article Title: miRNA-503 inhibition exerts anticancer effects and reduces tumor growth in mesothelioma

doi: 10.1186/s13046-025-03283-0

Figure Lengend Snippet: Histological analysis of miR-503 target genes and transcriptomic analysis of miR-503 inhibition. A Evaluation of the expression of BTG1, CCNG1, EDG1 and TIMP2, in normal pleural (PL) and pleural mesothelioma tissues (EPM epithelial istotype; SPM, sacomatoid istotype). Yellow Scale bar = 100 μm. Data shown are mean ± S.D. of five independent experiments. B IHC analysis of the putative miR-503 target genes on mice tumors treated with LNPs-anti-miR-503 or empty LNPs (original magnification 20X). A and B confirm the in vitro results. C Transcriptome analysis in MSTO cells after miR-503 inhibition detected about 500 deregulated genes (red upregulated, green downregulated). D Expression analysis of MM biomarkers deregulated by inhibition of miR-503 in MM cells. E q-PCR analysis in human MM samples of the miR-503 MM biomarkers confirms the inverse expression only for CXCL8, SERPINE1 and SPP. F IHC analysis of the miR-503 deregulated MM biomarkers on mice tumors treated with LNPs-anti-miR-503 or empty LNPs (original magnification 20X). For each IHC panel bar plots report the score for the samples analysed. Data presented as the mean ± SD of at least three independent experiments ( n = 3) for the in vitro experiments while for the in vivo analysis numbers of samples are reported in Methods. * p < 0.05, ** p < 0.01 *** p < 0.001 **** p < 0.0001 vs. control

Article Snippet: The primary antibodies used were: Rabbit anti-human Ki67 (DAKO Agilent, Santa Clara, CA USA), Rabbit anti-human BTG1 (orb35408 Biorbyt, Durham, NC, USA), Rabbit anti-human CCNG1 (orb167206 Biorbyt), Rabbit anti-human S1PR1 (EDG1) (orb350684 Biorbyt), Rabbit anti-human TIMP2 (orb543218 Biorbyt), Rabbit anti-human CXCL8 (17038-1-AP Proteintech Rosemont, IL, USA), Rabbit anti-human Osteopontin, (SPP 20416-1-AP Proteintech), Rabbit anti-human SERPINE1 (PAI-1 #49536 Cell Signaling, Danvers, MA, USA).

Techniques: Inhibition, Expressing, In Vitro, In Vivo, Control