Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE 2 production

doi: 10.1186/s12964-024-01851-z

Figure Lengend Snippet: Spns2/S1P deficiency enhances AA metabolism through p38-MAPK mediated cPLA 2 activation (A) The volcano plot illustrates the identification of 98 distinct metabolites between WT and Spns2 −/− PMs. AA and its derivatives are enriched in Spns2 −/− PMs. (B) KEGG enrichment analysis indicates elevated activities of glycerophospholipid metabolism and AA metabolism in Spns2 −/− PMs. (C) The heat map of the differential metabolites highlights the accumulation of AA derivatives and lysophospholipids in Spns2 −/− PMs. N = 6 biological replicates ( A to C ). ( D , E ) Deficient Spns2/S1P signaling enhances p38-MAPK mediated cPLA 2 activation. N = 3 biological replicates. Data are presented as mean ± s.e.m. (E) . P values were determined by one-way ANOVA with Sidak’s correction for multiple comparisons (E) . * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant

Article Snippet: PMs were pre-treated with the following compounds for 1-h before analysis, unless otherwise stated: 5 μM Spns2 inhibitor SLF1081851 (MedChemExpress, HY-149004), 1 μM S1P, 100 nM PF-04418948, 100 nM ONO-AE3-208, 200 nM PGE 2 (TargetMOI, T5014), 2 μM S1PR1 antagonist W146 (Cayman Chemical, 10009109), 2 μM S1PR2 antagonist JTE013 (Cayman Chemical, 10009458), 2 μM S1PR3 antagonist TY-52,156 (TargetMOI, T17183), 2 μM S1PR4 antagonist CYM50358 (Tocris Bioscience, 4679), 5 μM S1PR1 agonist CYM5442 (TargetMOI, T2026), 5 μM S1PR2 agonist CYM5520 (TargetMOI, T22703), 5 μM S1PR3 agonist CYM5541 (TargetMOI, T3961), 5 μM S1PR4 agonist CYM50260 (TargetMOI, T15031), and 50 nM S1PR5 agonist A971432 (Cayman Chemical, 25326).

Techniques: Activation Assay

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE 2 production

doi: 10.1186/s12964-024-01851-z

Figure Lengend Snippet: Spns2/S1P signaling impacts mitochondrial functions through coordinated activation of multiple S1P receptors (A) All five S1PRs are transcriptionally detectable in PMs. N = 3 biological replicates. (B) Inhibition of individual S1PRs in WT PMs increases the gene expression of Ptges , while activation of S1PR2 and S1PR4 in Spns2 −/− PMs may slightly reduce Ptges expression. N = 4 biological replicates. (C) In WT PMs, blocking individual S1PRs reduces the fluorescence intensity of MitoTracker™ Red, indicating diminished ΔΨm. (D) S1PR3 blockade significantly increases the fluorescence intensity of CytoFix™ MitoRed, indicating increased mitochondrial mass, while blockade of other receptors may cause a slight increase in mitochondrial mass. (E) All treatments result in reduced average Δψm (calculated by the ratio of MT/CF) in WT PMs. N = 3 biological replicates ( C to E ). ( F , G ) In Spns2 −/− PMs, activation of S1PR3 and S1PR5 increases MitoTracker™ Red fluorescence intensity (F) , but none of the treatments affect mitochondrial mass (G) . (H) Only S1PR3 activation partially restores the average Δψm in Spns2 −/− PMs. N = 3 biological replicates ( F to H ). (I) All the antagonists increase mtROS generation in WT PMs, especially with S1PR2 and S1PR4 blockade. N = 3 biological replicates. (J) In Spns2 −/− PMs, activation of S1PR2 and S1PR4 attenuates oxidative stress. N = 3 biological replicates. Data are presented as mean ± s.e.m. P values were determined by unpaired t -test (A) and one-way ANOVA with Sidak’s correction for multiple comparisons ( B to J ). * P < 0.05; ** P < 0.01; *** P < 0.001; n.s., not significant

Article Snippet: PMs were pre-treated with the following compounds for 1-h before analysis, unless otherwise stated: 5 μM Spns2 inhibitor SLF1081851 (MedChemExpress, HY-149004), 1 μM S1P, 100 nM PF-04418948, 100 nM ONO-AE3-208, 200 nM PGE 2 (TargetMOI, T5014), 2 μM S1PR1 antagonist W146 (Cayman Chemical, 10009109), 2 μM S1PR2 antagonist JTE013 (Cayman Chemical, 10009458), 2 μM S1PR3 antagonist TY-52,156 (TargetMOI, T17183), 2 μM S1PR4 antagonist CYM50358 (Tocris Bioscience, 4679), 5 μM S1PR1 agonist CYM5442 (TargetMOI, T2026), 5 μM S1PR2 agonist CYM5520 (TargetMOI, T22703), 5 μM S1PR3 agonist CYM5541 (TargetMOI, T3961), 5 μM S1PR4 agonist CYM50260 (TargetMOI, T15031), and 50 nM S1PR5 agonist A971432 (Cayman Chemical, 25326).

Techniques: Activation Assay, Inhibition, Gene Expression, Expressing, Blocking Assay, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE 2 production

doi: 10.1186/s12964-024-01851-z

Figure Lengend Snippet: Schematic illustration of how Spns2/S1P signaling modulates mitochondrial functions and inflammatory response through PGE 2 production in macrophages

Article Snippet: PMs were pre-treated with the following compounds for 1-h before analysis, unless otherwise stated: 5 μM Spns2 inhibitor SLF1081851 (MedChemExpress, HY-149004), 1 μM S1P, 100 nM PF-04418948, 100 nM ONO-AE3-208, 200 nM PGE 2 (TargetMOI, T5014), 2 μM S1PR1 antagonist W146 (Cayman Chemical, 10009109), 2 μM S1PR2 antagonist JTE013 (Cayman Chemical, 10009458), 2 μM S1PR3 antagonist TY-52,156 (TargetMOI, T17183), 2 μM S1PR4 antagonist CYM50358 (Tocris Bioscience, 4679), 5 μM S1PR1 agonist CYM5442 (TargetMOI, T2026), 5 μM S1PR2 agonist CYM5520 (TargetMOI, T22703), 5 μM S1PR3 agonist CYM5541 (TargetMOI, T3961), 5 μM S1PR4 agonist CYM50260 (TargetMOI, T15031), and 50 nM S1PR5 agonist A971432 (Cayman Chemical, 25326).

Techniques:

Journal: Cardiovascular Research

Article Title: Extracellular component hyaluronic acid and its receptor Hmmr are required for epicardial EMT during heart regeneration

doi: 10.1093/cvr/cvv190

Figure Lengend Snippet: FAK and Src inhibition suppressed angiogenesis in regenerating hearts. (A–C) At 30 dpa, hearts injected for 10 days with PF-573228, a FAK inhibitor (B) (n = 10), or with SKI-1 (C) (n = 13), a Src inhibitor, failed to properly regenerate the heart compared with DMSO controls (A) (n = 31). Red arrows delineate scar. (D) Graph showing clot area at 30 dpa after injections of PF-573228 or SKI-1. (E–M) PF-573228 and SKI-1 injections inhibited angiogenesis. (E–G) AFOG staining of hearts at 10 dpa, injected with DMSO (E) (n = 15), PF-573228 (F) (n = 10), or SKI-1 (G) (n = 10). (H–M) Tg(fli1a:EGFP) hearts injected with DMSO (H and K), PF-573228 (I and L), or with SKI-1 (J and M), immunostained for MHC to detect resection plane. White arrows indicate new vessels formed inside the clot. (N) Quantification fli1a+:EGFP area in the clot at 10 dpa. FAK and Src inhibition significantly reduced new vessel formation. Black boxes delimitate the areas of confocal imaging. Black bars in graphs indicate the mean and error bars the SEM. Scale bars, 100 μm. *P < 0.05; **P < 0.01; ***P < 0.001 one-way ANOVA.

Article Snippet: To suppress Src Kinase, 3 µL of 200 µM Src Inhibitor 1 (SKI-1) (Sigma) or vehicle DMSO was injected.

Techniques: Inhibition, Injection, Staining, Imaging

Journal: Cardiovascular Research

Article Title: Extracellular component hyaluronic acid and its receptor Hmmr are required for epicardial EMT during heart regeneration

doi: 10.1093/cvr/cvv190

Figure Lengend Snippet: HA and its receptor Hmmr are necessary for epicardial cell migration in ex vivo culture. (A–D) Ex vivo epicardial migration assay showing decreased Wt1b:EGFP+ migratory behaviour after hmmrVMO (B) (n = 8) and PF-573228 (D) (n = 7) treatments compared with controls (A) (n = 14) and (C) (n = 7). Images were captured at 3 days post-extraction of the heart. The migration of Wt1b:EGFP+ epicardial cells (red arrows) was measured from Day 1 to Day 3. In control PBS, DMSO, or hmmr-MutMO treatments, epicardial cells migrated on the fibrin-coated plates. In contrast, treatment with hmmrVMO, PF-573228 or SKI-1 significantly suppressed epicardial cell migration. (E–H) Graphs showing migration of Wt1b:EGFP+ cells under various treatments, including suppressing HA production in the hearts with HMC (E) (n = 13), hmmrVMO (F) (n = 8), PF-573228 (G) (n = 7), and with SKI-1, the Src kinase inhibitor (H) (n = 8). Black bars in graphs indicate the mean and error bars the SEM. Scale bars, 250 μm. *P < 0.05; **P < 0.01; ***P < 0.001. Two-way ANOVA.

Article Snippet: To suppress Src Kinase, 3 µL of 200 µM Src Inhibitor 1 (SKI-1) (Sigma) or vehicle DMSO was injected.

Techniques: Migration, Ex Vivo

Journal: Journal of Inflammation (London, England)

Article Title: Different roles for non-receptor tyrosine kinases in arachidonate release induced by zymosan and Staphylococcus aureus in macrophages

doi: 10.1186/1476-9255-3-8

Figure Lengend Snippet: Inhibition by PP2 or SKI-1 of zymosan- and S.aureus -induced release of arachidonate . Macrophages were labeled with [3H]arachidonic acid for 20 h. The cells were pretreated for 15 min with the indicated concentrations of PP2 (A) or SKI-1 (B) followed by stimulation with either zymosan (●) for 45 min or S.aureus (▲) for 60 min. Results are mean ± SEM (n = 3) and corrected for the release in control cultures.

Article Snippet: LFM-A13 and Src kinase inhibitor I (SKI-1) from Calbiochem (La Jolla, CA, USA).

Techniques: Inhibition, Labeling

Journal: Journal of Inflammation (London, England)

Article Title: Different roles for non-receptor tyrosine kinases in arachidonate release induced by zymosan and Staphylococcus aureus in macrophages

doi: 10.1186/1476-9255-3-8

Figure Lengend Snippet: Inhibition by PP2 or SKI-1 of zymosan- and S.aureus - induced phosphorylation of MAP kinases . Macrophages were pretreated for 15 min with PP2 (1–10 μM), followed by stimulation with zymosan ( A ) or S.aureus ( B ) for 20 min. ( C ) Macrophages were pretreated for 15 min with SKI-1 (5 μM), followed by stimulation with zymosan or S.aureus for 20 min. Equal amounts of cell lysate were run on 10% polyacrylamide gels and probed with phosphospecific antibodies against ERK, p38 and JNK. The membrane was reprobed with ERK-2 antibody to verify equal loading of protein.

Article Snippet: LFM-A13 and Src kinase inhibitor I (SKI-1) from Calbiochem (La Jolla, CA, USA).

Techniques: Inhibition

Journal: Journal of Inflammation (London, England)

Article Title: Different roles for non-receptor tyrosine kinases in arachidonate release induced by zymosan and Staphylococcus aureus in macrophages

doi: 10.1186/1476-9255-3-8

Figure Lengend Snippet: Zymosan but not S.aureus induced tyrosine phosphorylation of PLC γ2 . ( A ) Macrophages were stimulated with zymosan(z) or S.aureus ( S.a ) for 45 min. (B and C) Macrophages were pretreated for 15 min with either PP2 (5 μM), wortmannin (W, 100 nM) ( B ) or SKI-1 (5 μM) ( C ) followed by stimulation with zymosan for 30 min. Cell lysates were immunoprecipitated with antibody against PLCγ2 as described, followed by Western blot analysis with phosphotyrosine-specific antibody. The membrane was stripped and reprobed with antibody against PLCγ2. The data are representative of three separate experiments.

Article Snippet: LFM-A13 and Src kinase inhibitor I (SKI-1) from Calbiochem (La Jolla, CA, USA).

Techniques: Immunoprecipitation, Western Blot

Journal: Journal of Inflammation (London, England)

Article Title: Different roles for non-receptor tyrosine kinases in arachidonate release induced by zymosan and Staphylococcus aureus in macrophages

doi: 10.1186/1476-9255-3-8

Figure Lengend Snippet: Zymosan and bacteria but not LPS or peptidoglycan (PGN) induce phosphorylation of Btk . ( A ) Macrophages were stimulated with zymosan (zym), S.aureus ( S.a .), LPS or PGN for 30 min. (B and C) Macrophages were pretreated for 15 min with either wortmannin (W, 100 nM), PP2 (5 μM), SU6656 (5 μM) or SKI-1 (5 μM), followed by stimulation with zymosan ( B ) or S.aureus ( C ) for 30 min. Equal amounts of cell lysate were run on polyacrylamide gels and probed with phosphospecific antibodies against Btk. The membrane was reprobed with ERK-2 antibody to verify equal loading of proteins. The data are representative of three separate experiments.

Article Snippet: LFM-A13 and Src kinase inhibitor I (SKI-1) from Calbiochem (La Jolla, CA, USA).

Techniques:

Journal: The Journal of Cell Biology

Article Title: Adhesion to fibronectin regulates Hippo signaling via the FAK–Src–PI3K pathway

doi: 10.1083/jcb.201501025

Figure Lengend Snippet: PI3K, PDK1, and Src regulation of nuclear YAP via Lats in serum-starved, subconfluent cells. (A) PI3K and PDK1 inhibitors relative to Lats. MCF-10A cells transfected with control, Nf2, or Lats1/2 siRNAs were serum starved and treated with DMSO (solvent control), 10 µM wortmannin (PI3K inhibitor), or 5 µM BX-795 (PDK1 inhibitor) for 30 min. On-target plus nontargeting pool was used as a control siRNA. Localization of endogenous YAP was identified by immunofluorescence staining. (B) SFK inhibitors and YAP localization. Serum-starved, low cell density MCF-10A cells were incubated with SFK inhibitors (10 µM each of PP2, dasatinib, SKI-1, and SU6656) for 30 min. 10 µM each of PP3 and imatinib were used as controls. YAP subcellular localization was determined by immunofluorescence staining. Alexa Fluor 594 secondary antibody was used for SU6656, which has high background green fluorescence. (C) Biochemical effects of Src inhibition. PP3- or PP2-treated MCF-10A cells were analyzed by Western blot using anti-YAP and anti–phospho-YAP (S127) antibodies. Phosphorylated YAP was detected by mobility shift on Phos-tag SDS-PAGE. (D) SFK inhibitors relative to Lats. MCF-10A cells transfected with control, Nf2, or Lats1/2 siRNA were serum starved and treated with 10 µM PP3 or PP2. After 30 min, cells were fixed for immunofluorescence staining with anti-YAP antibody. (E) Depletion of individual SFK. MCF-10A cells were transfected with control, Src, Fyn, or Yes siRNA. After serum starvation, subcellular localization of endogenous YAP was identified by immunofluorescence staining and quantified based on the criteria shown under the graph. More than 120 cells from four random views were quantified. (F) Src knockdown relative to Lats. MCF-10A cells were transfected with control, Src, Lats1/2, or combined siRNA of Src and Lats1/2. Cells were serum starved for 24 h before fixation and stained with anti-YAP antibody. (A, B, and D–F) One of three independent results is presented. Bars, 25 µm.

Article Snippet: The following inhibitors were used: SFK inhibitors SKI-1 and SU6656, Src/Abl inhibitor dasatinib monohydrate, Abl inhibitor imatinib mesylate, PDK1 inhibitor BX-795, FAK inhibitors PF-573228 and PF-562271 (Santa Cruz Biotechnology, Inc.), SFK inhibitor PP2 and its inactive analogue PP3 (EMD Millipore), and PI3K inhibitor wortmannin (Sigma-Aldrich).

Techniques: Transfection, Control, Solvent, Immunofluorescence, Staining, Incubation, Fluorescence, Inhibition, Western Blot, Mobility Shift, SDS Page, Knockdown

Journal: Stem cell research

Article Title: Src-family Tyrosine Kinase Activities are Essential for Differentiation of Human Embryonic Stem Cells

doi: 10.1016/j.scr.2014.09.007

Figure Lengend Snippet: A) The hES cell lines H1, H7 and H9 were maintained under feeder-free conditions on Matrigel-coated plates using mTeSR1 medium. All three lines grew as large domed colonies with well-circumscribed boundaries. B) H1 hES cells were harvested 5 days after passage for RNA isolation. SFK (Blk, Fgr, Fyn, Hck, Lck, Lyn, c-Src, c-Yes), Src-related kinase (Brk, Frk, Srm) and pluripotency marker (Oct4) expression were determined by RT-PCR with GAPDH as a positive control. Images of agarose gels of the PCR products are shown after 30 and 35 PCR cycles. C) Relative SFK (Fyn, Hck, Lck, Lyn, c-Src, c-Yes) and pluripotency marker (Oct4, Nanog) expression levels across three hES cell lines. Transcript levels for the indicated genes were determined by quantitative real-time RT-PCR for the H1, H7 and H9 hES cell lines. The results are expressed as the average fold-change in mRNA level in H7 and H9 cells relative to H1 cells ± SEM (n = 3, *p < 0.05; pairwise xed reallocation randomization test). SFK expression varied by less than 2-fold across these hES cell lines (gray area), with the exception of Hck expression in H7 cells.

Article Snippet: The SFK inhibitors SKI-1 and PP2 were purchased from EMD-Millipore-Calbiochem while A-419259 was purchased from Santa Cruz Biotechnology.

Techniques: Isolation, Marker, Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Quantitative RT-PCR

Journal: Stem cell research

Article Title: Src-family Tyrosine Kinase Activities are Essential for Differentiation of Human Embryonic Stem Cells

doi: 10.1016/j.scr.2014.09.007

Figure Lengend Snippet: Lysates were prepared from mouse ES cells (D3 line), human ES cells (H1 and H7 lines) and 6-day EBs derived from them. Endogenous SFKs (c-Src, c-Yes, Fyn, Lck, Hck, Lyn) were immunoprecipitated and blotted with a phosphospecific antibody for the active form of each kinase (pY418 antibody, which recognizes the conserved phosphotyrosine residue in the activation loop of the kinase domain) as well as the respective SFK protein. Cell lysates were also blotted for Oct4 as marker of ES cell self-renewal plus actin as a loading control. This experiment was repeated twice with comparable results; a representative example is shown.

Article Snippet: The SFK inhibitors SKI-1 and PP2 were purchased from EMD-Millipore-Calbiochem while A-419259 was purchased from Santa Cruz Biotechnology.

Techniques: Derivative Assay, Immunoprecipitation, Residue, Activation Assay, Marker, Control

Journal: Stem cell research

Article Title: Src-family Tyrosine Kinase Activities are Essential for Differentiation of Human Embryonic Stem Cells

doi: 10.1016/j.scr.2014.09.007

Figure Lengend Snippet: Self-renewing hES cells (H1 and H7 lines) were cultured in the absence or presence of A-419259 (1.0 μM) for 16 h, followed by lysis and immunoprecipitation of c-Src and Lck. Each immunoprecipitate was then blotted with a phosphospecific antibody for the active form of each kinase (pY418 antibody, which recognizes the conserved phosphotyrosine residue in the activation loop of the kinase domain) as well as the respective SFK protein. Cell lysates were also blotted for pY418 as well as Oct4 as a marker of ES cell self-renewal. This experiment was repeated twice with comparable results; a representative example is shown.

Article Snippet: The SFK inhibitors SKI-1 and PP2 were purchased from EMD-Millipore-Calbiochem while A-419259 was purchased from Santa Cruz Biotechnology.

Techniques: Cell Culture, Lysis, Immunoprecipitation, Residue, Activation Assay, Marker

Journal: Stem cell research

Article Title: Src-family Tyrosine Kinase Activities are Essential for Differentiation of Human Embryonic Stem Cells

doi: 10.1016/j.scr.2014.09.007

Figure Lengend Snippet: The H7 line of hES cells was grown in mTeSR self-renewal medium or switched to differentiation medium in the absence (Control) or presence of A-419259 (1 μM). Cells were fixed, permeabilized and immunostained for the SFK Lck and the nuclear pluripotency factor Oct4 four days later. DAPI-stained nuclei are also shown. The merged image combines cells in the Lck and Oct4 panels only. This experiment was repeated three times with comparable results; a representative field of cells is shown for each condition.

Article Snippet: The SFK inhibitors SKI-1 and PP2 were purchased from EMD-Millipore-Calbiochem while A-419259 was purchased from Santa Cruz Biotechnology.

Techniques: Control, Staining