Journal: Scientific Reports

Article Title: Essential Roles of RNA-binding Protein HuR in Activation of Hepatic Stellate Cells Induced by Transforming Growth Factor-β1

doi: 10.1038/srep22141

Figure Lengend Snippet: The correlation between mRNA levels of HuR and α-SMA ( a ), Col α1(I) ( b ), TGF-β1 ( c ) or SphK1 ( d ) in mouse fibrotic livers. ( e ) Liver non-parenchymal cells of Sham or BDL mice were isolated and RIP analysis was performed. SphK1 mRNA was measured by qRT-PCR, and the PCR products were size-fractionated in a 2% agarose gel. The gels are run under the same experimental conditions. ( f ) Liver non-parenchymal cells of mice treated with OO or CCl 4 were isolated and RIP analysis was performed as described.

Article Snippet: Probes (Applied Biosystems) were as follows: human SphK1: hs00184211_m1; mouse SphK1:Mm00448841_g1.

Techniques: Isolation, Quantitative RT-PCR, Agarose Gel Electrophoresis

Journal: Scientific Reports

Article Title: Essential Roles of RNA-binding Protein HuR in Activation of Hepatic Stellate Cells Induced by Transforming Growth Factor-β1

doi: 10.1038/srep22141

Figure Lengend Snippet: LX-2 were pre-treated with LMB (10 nmol/L) for 2 hours and followed by TGF-β1 (10 ng/mL) treatment for 24 hours. HuR levels in cytoplasmic or nuclear lysates ( a ), and total HuR, SphK1, α-SMA and Col α1(I) levels ( b ) were evaluated by Western blot analysis. The cropped blots are used in the figure and full-length blots are presented in . The levels of β-Tubulin (a cytoplasmic protein) and HDAC1 (a nuclear protein) in the same samples were assessed to ascertain the quality of the fractionation procedure and to detect loading differences. The intensity of each band was quantified and normalized to β-Tubulin. The values were the mean intensity normalized of each band (fold over basal). ( c ) HuR cytoplasmic accumulation was evaluated by immunofluorescence. DAPI was used to visualize nuclei (blue). ( d ) SphK1, α-SMA or Col α1(I) mRNA expression was examined by qRT-PCR analysis. Data are presented as the means ± SEM derived from at least three independent experiments. * P < 0.05, versus untreated control cells. † P < 0.05, versus cells treated with TGF-β1 alone. Scale bars = 25 μm.

Article Snippet: Probes (Applied Biosystems) were as follows: human SphK1: hs00184211_m1; mouse SphK1:Mm00448841_g1.

Techniques: Western Blot, Fractionation, Immunofluorescence, Expressing, Quantitative RT-PCR, Derivative Assay, Control

Journal: Scientific Reports

Article Title: Essential Roles of RNA-binding Protein HuR in Activation of Hepatic Stellate Cells Induced by Transforming Growth Factor-β1

doi: 10.1038/srep22141

Figure Lengend Snippet: ( a,b ) LX-2 or primary mouse HSCs were transfected with SCR siRNA or HuR siRNA. 48 hours later, cells were treated with TGF-β1 for another 24 hours. ( a ) The mRNA expression of HuR was detected to confirm the efficiency of HuR knockdown. ( b ) SphK1 mRNA level was also evaluated. ( c ) Cells were transfected with pcDNA or pcDNA-flag-HuR plasmids. 48 hours later, qPCR was performed to assess HuR or SphK1 mRNA level, respectively. ( d,e ) Cells described in were collected and protein levels of HuR ( d ) and SphK1 ( e ) were evaluated. ( f,g ) Cells described in were collected and protein level of HuR ( f ) or SphK1 ( g ) was evaluated. The cropped blots are used in the figure and full-length blots are presented in . The values were the mean intensity normalized of each band (fold over basal). Data are presented as the means ± SEM derived from at least three independent experiments. * P < 0.05, versus untreated control cells. † P < 0.05, versus cells treated with TGF-β1 alone.

Article Snippet: Probes (Applied Biosystems) were as follows: human SphK1: hs00184211_m1; mouse SphK1:Mm00448841_g1.

Techniques: Transfection, Expressing, Knockdown, Derivative Assay, Control

Journal: Scientific Reports

Article Title: Essential Roles of RNA-binding Protein HuR in Activation of Hepatic Stellate Cells Induced by Transforming Growth Factor-β1

doi: 10.1038/srep22141

Figure Lengend Snippet: ( a ) LX-2 treated with or without TGF-β1 was collected. RIP experiment was performed as described in methods. SphK1 mRNA was measured by qRT-PCR, and the PCR products were size-fractionated in a 2% agarose gel. LX-2 were transfected with SCR siRNA ( b ) or HuR siRNA ( c ) before TGF-β1 treatment for 24 hours, the half-life of SphK1 mRNA was determined by ActD pulse-chase experiments. LX-2 was pre-treated without ( d ) or with ( e ) LMB for 2 hours before TGF-β1 treatment, and SphK1 mRNA half-life was examined. ( f ) Cells were transfected with pcDNA-flag-HuR plasmids and the half-life of SphK1 mRNA was tested. Data are presented as the means ± SEM derived from at least three independent experiments.

Article Snippet: Probes (Applied Biosystems) were as follows: human SphK1: hs00184211_m1; mouse SphK1:Mm00448841_g1.

Techniques: Quantitative RT-PCR, Agarose Gel Electrophoresis, Transfection, Pulse Chase, Derivative Assay

Journal: Scientific Reports

Article Title: Essential Roles of RNA-binding Protein HuR in Activation of Hepatic Stellate Cells Induced by Transforming Growth Factor-β1

doi: 10.1038/srep22141

Figure Lengend Snippet: ( a ) LX-2 was treated with SKI before transfected with pcDNA-flag-HuR plasmids, α-SMA and Col α1(I) protein expressions were evaluated. The cropped blots are used in the figure and full-length blots are presented in . Cells were co-transfected with SphK1 siRNA and pcDNA-flag-HuR plasmids, mRNA ( b ) and protein ( c ) levels of SphK1 were examined to confirm the efficiency of SphK1 knockdown. α-SMA and Col α1(I) protein expressions ( d ) were detected. The cropped blots are used in the figure and full-length blots are presented in . β-Tubulin levels were assessed as a loading control. The values were the mean intensity normalized of each band (fold over basal). Data are presented as the means ± SEM derived from at least three independent experiments. * P < 0.05, versus the control cells.

Article Snippet: Probes (Applied Biosystems) were as follows: human SphK1: hs00184211_m1; mouse SphK1:Mm00448841_g1.

Techniques: Transfection, Knockdown, Control, Derivative Assay

Journal: Scientific Reports

Article Title: Essential Roles of RNA-binding Protein HuR in Activation of Hepatic Stellate Cells Induced by Transforming Growth Factor-β1

doi: 10.1038/srep22141

Figure Lengend Snippet: LX-2 were transfected with specific siRNA of HuR or SphK1, or pre-incubated with SphK1 inhibitor SKI before TGF-β1 treatment. mRNA levels of α-SMA and Col α1(I) in response to TGF-β1 in the presence of HuR siRNA ( a ), SKI ( b ), or SphK1 siRNA ( c ) were evaluated by qRT-PCR. Protein expressions of α-SMA and Col α1(I) in response to TGF-β1 in the presence of HuR siRNA ( d ), SKI ( e ), or SphK1 siRNA ( f ) were detected by Western blot. The cropped blots are used in the figure and full-length blots are presented in . β-Tubulin levels were assessed as a loading control. The values were the mean intensity normalized of each band (fold over basal). ( g ) Primary mouse HSCs were transfected with specific siRNA of HuR before TGF-β1 treatment. mRNAs of α-SMA and Col α1(I) were observed. ( h ) Scheme of HSCs activation. Data are presented as the means ± SEM derived from at least three independent experiments. * P < 0.05, versus the control cells without treatment. † P < 0.05, versus cells treated with TGF-β1 alone.

Article Snippet: Probes (Applied Biosystems) were as follows: human SphK1: hs00184211_m1; mouse SphK1:Mm00448841_g1.

Techniques: Transfection, Incubation, Quantitative RT-PCR, Western Blot, Control, Activation Assay, Derivative Assay

Journal: Nature communications

Article Title: N-AS-triggered SPMs are direct regulators of microglia in a model of Alzheimer's disease.

doi: 10.1038/s41467-020-16080-4

Figure Lengend Snippet: Fig. 2 N-AS-acetylated COX2 produces SPMs. a Human recombinant COX2 treated in the presence or absence of 500 μM N-AS or aspirin was incubated with AA, EPA, or DHA in presence of human 5-LOX, and then SPM precursors were identified using systematic LC-MS/MS. Representative chromatogram showing the SPM precursors (Top, 15-HETE; Middle, 18-HEPE; Bottom, 17-HDHA). b Human recombinant COX2 treated in the presence of 500 μM N-AS or aspirin was incubated with AA, EPA, or DHA in presence of human 5-LOX. Related MS/MS spectra employed for identification of SPMs. 15R-LXA4, RvE1, and RvD1. c Quantification of 15R-LXA4, RvE1, and RvD1 in COX2 WT and COX2 S565A treated with 5-LOX in presence of N-AS, aspirin or not (n = 5–7 independent experiments per group). c One-way analysis of variance, Tukey’s post hoc test. All error bars indicate s.e.m. Source data are provided as a Source data file.

Article Snippet: Single point mutations in SphK1 (R56A, R57A, R24A, R185A, D178A, and F192A) and COX2 (S565A, N181A, T564A, and S567A) were generated using the In-Fusion Cloning Kits (Clontech) and ORF cDNA expression plasmids of human SphK1 and COX2 gene (Sino Biological Inc, HG15679-NH and HG12036-NH), according to the manufacturer’s instructions.

Techniques: Recombinant, Incubation, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy

Journal: Journal of translational medicine

Article Title: Targeting the SphK1/S1P/PFKFB3 axis suppresses hepatocellular carcinoma progression by disrupting glycolytic energy supply that drives tumor angiogenesis.

doi: 10.1186/s12967-023-04830-z

Figure Lengend Snippet: Fig. 1 PF-543 inhibits SphK1 activity in DEN-treated mice. A Total and phospho-SphK1 levels were examined using immunohistochemical staining in tumorous (T) and para-tumorous (para-T) tissues of human HCC specimens; scale bar = 50 μm; n = 10. B Schematic illustration of treatment schedules for in vivo studies, created with BioRender.com. C Body weight over 12 weeks of vehicle (veh) or PF-543 (PF) treatment. D Liver mass was weighed. E phospho(p)-SphK1 SphK1 and SphK2 protein levels in liver tissues were determined using Western blotting. F Hepatic levels of ceramide (Cer), sphingosine (Sph), and S1P were analyzed using lipidomics. C-F n = 9. Data are expressed as mean ± SD. *p < 0.05; ***p < 0.001

Article Snippet: Immunohistochemistry (IHC) and immunofluorescent (IF) staining were conducted on paraffin-embedded sections and cryosections, respectively, using antibodies against SphK1 (Santa Cruz Biotechnology), phospho-SphK1 (Proteintech), Ki67 (Abcam), CD31 (CST for IHC and BD Pharmingen for IF), cleaved caspase-3 (Cell Signaling Technology, CST) and PFKFB3 (Proteintech).

Techniques: Activity Assay, Immunohistochemical staining, Staining, In Vivo, Western Blot

Journal: Journal of translational medicine

Article Title: Targeting the SphK1/S1P/PFKFB3 axis suppresses hepatocellular carcinoma progression by disrupting glycolytic energy supply that drives tumor angiogenesis.

doi: 10.1186/s12967-023-04830-z

Figure Lengend Snippet: Fig. 2 Inhibition of SphK1 suppresses HCC progression in DEN-treated mice. DEN-injected mice were treated with vehicle (veh) or PF-543 (PF) for 12 weeks. A The number and maximal diameter of visible liver tumors were quantified from macroscopic images. B The number and maximal diameter of intrahepatic liver tumors were quantified from the scanning of the entire H&E-stained liver tissue sections. C Cell proliferation in non-tumorous (NT) and tumorous (T) liver tissues was stained and quantified using Ki67 immunohistochemistry; scale bar = 50 μm. Data are expressed as mean ± SD. n = 9. *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: Immunohistochemistry (IHC) and immunofluorescent (IF) staining were conducted on paraffin-embedded sections and cryosections, respectively, using antibodies against SphK1 (Santa Cruz Biotechnology), phospho-SphK1 (Proteintech), Ki67 (Abcam), CD31 (CST for IHC and BD Pharmingen for IF), cleaved caspase-3 (Cell Signaling Technology, CST) and PFKFB3 (Proteintech).

Techniques: Inhibition, Injection, Staining, Immunohistochemistry

Journal: Journal of translational medicine

Article Title: Targeting the SphK1/S1P/PFKFB3 axis suppresses hepatocellular carcinoma progression by disrupting glycolytic energy supply that drives tumor angiogenesis.

doi: 10.1186/s12967-023-04830-z

Figure Lengend Snippet: Fig. 3 SphK1 inhibition or ablation reduces vessel density in HCC tumors. A DEN-injected mice were treated with vehicle (veh) or PF-543 (PF) for 12 weeks. Blood vessels in non-tumorous (NT) and tumorous (T) liver tissues were stained and quantified by CD31 immunohistochemistry; scale bar = 50 μm; n = 9. B Blood vessels in DEN-injected wild-type (WT) and Sphk1 knockout (KO) mice were examined by CD31 immunohistochemistry; scale bar = 50 μm; n = 5. Data are expressed as mean ± SD. **p < 0.01; ***p < 0.001

Article Snippet: Immunohistochemistry (IHC) and immunofluorescent (IF) staining were conducted on paraffin-embedded sections and cryosections, respectively, using antibodies against SphK1 (Santa Cruz Biotechnology), phospho-SphK1 (Proteintech), Ki67 (Abcam), CD31 (CST for IHC and BD Pharmingen for IF), cleaved caspase-3 (Cell Signaling Technology, CST) and PFKFB3 (Proteintech).

Techniques: Inhibition, Injection, Staining, Immunohistochemistry, Knock-Out

Journal: Journal of translational medicine

Article Title: Targeting the SphK1/S1P/PFKFB3 axis suppresses hepatocellular carcinoma progression by disrupting glycolytic energy supply that drives tumor angiogenesis.

doi: 10.1186/s12967-023-04830-z

Figure Lengend Snippet: Fig. 4 Inhibition of SphK1 impairs angiogenesis in HUVECs. Primary HUVECs were treated with PF-543 at the indicated concentrations for 16 h. A and B Tube formation was induced by 50 ng/ml VEGF-A (A) or conditioned medium collected from Huh7 HCC cell culture (B). Quantification of tube formation is presented as the number of junctions, segment length, and total branching length. kpx, 1000 pixels; scale bar = 200 μm (A) or 100 μm (B); n = 3. C Sprouting assays were performed in three-dimensional spheroids. Quantitation of the sprouting is presented as cumulative sprout length and number of sprouts per spheroid; scale bar = 100 μm; n = 10. D Cell migration was determined by transwell assay, and migrated cells were stained with crystal violet; scale bar = 100 μm; n = 3. E Cell viability was determined using MTS assay; n = 4. F Cell death was assessed using flow cytometry with propidium iodide (PI) staining; PI-, living cells (green); PI+, dead cells (red); n = 3. Data are expressed as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: Immunohistochemistry (IHC) and immunofluorescent (IF) staining were conducted on paraffin-embedded sections and cryosections, respectively, using antibodies against SphK1 (Santa Cruz Biotechnology), phospho-SphK1 (Proteintech), Ki67 (Abcam), CD31 (CST for IHC and BD Pharmingen for IF), cleaved caspase-3 (Cell Signaling Technology, CST) and PFKFB3 (Proteintech).

Techniques: Inhibition, Cell Culture, Quantitation Assay, Migration, Transwell Assay, Staining, MTS Assay, Flow Cytometry

Journal: Journal of translational medicine

Article Title: Targeting the SphK1/S1P/PFKFB3 axis suppresses hepatocellular carcinoma progression by disrupting glycolytic energy supply that drives tumor angiogenesis.

doi: 10.1186/s12967-023-04830-z

Figure Lengend Snippet: Fig. 5 SphK1 promotes angiogenesis by regulating the glycolytic modulator PFKFB3. A Primary HUVECs were treated with 5 µM PF-543 for 16 h, prior to stimulation with 50 ng/ml VEGF-A for 15 min. B Following 1 h pre-treatment with 10 µM MG-132, primary HUVECs were treated with PF-543 at 5 µM for 16 h. A and B Western blotting analyses of the indicated proteins; n = 3. C and D Glycolytic rate, capacity and reserve were determined using Seahorse real-time glycolytic stress assay in PF-543-treated (C) or shRNA-mediated SphK1 knockdown (D) primary HUVECs. ECAR, extracellular acidification rate; n = 5. Data are expressed as mean ± SD. *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: Immunohistochemistry (IHC) and immunofluorescent (IF) staining were conducted on paraffin-embedded sections and cryosections, respectively, using antibodies against SphK1 (Santa Cruz Biotechnology), phospho-SphK1 (Proteintech), Ki67 (Abcam), CD31 (CST for IHC and BD Pharmingen for IF), cleaved caspase-3 (Cell Signaling Technology, CST) and PFKFB3 (Proteintech).

Techniques: Western Blot, shRNA, Knockdown

Journal: Journal of translational medicine

Article Title: Targeting the SphK1/S1P/PFKFB3 axis suppresses hepatocellular carcinoma progression by disrupting glycolytic energy supply that drives tumor angiogenesis.

doi: 10.1186/s12967-023-04830-z

Figure Lengend Snippet: Fig. 7 Model depicting the anti-HCC actions of PF-543. Elevated pro-angiogenic factors, exemplified by VEGF-A, act through their receptors to drive sprouting angiogenesis in tumor endothelial cells (EC), promoting tumor neovascularization and HCC progression. PFKFB3 serves as a molecular switch in this process, dictating the glycolytic energy supply that is essential for sprouting angiogenesis. The selective SphK1 inhibitor PF-543 abrogates S1P production, subsequently turning off the PFKFB3-mediated glycolytic switch, which leads to the inhibition of sprouting angiogenesis and eventually suppression of HCC progression. The graphical abstract was created with BioRender.com. . The 3D conformer of PF-543 was adapted from PubChem, pubchem.ncbi.nlm.nih.gov, CID 66,577,038

Article Snippet: Immunohistochemistry (IHC) and immunofluorescent (IF) staining were conducted on paraffin-embedded sections and cryosections, respectively, using antibodies against SphK1 (Santa Cruz Biotechnology), phospho-SphK1 (Proteintech), Ki67 (Abcam), CD31 (CST for IHC and BD Pharmingen for IF), cleaved caspase-3 (Cell Signaling Technology, CST) and PFKFB3 (Proteintech).

Techniques: Inhibition

Journal:

Article Title: Extracellular export of sphingosine kinase-1a contributes to the vascular S1P gradient

doi: 10.1042/BJ20060251

Figure Lengend Snippet: (A) Amino acid sequence alignment of hSphk1 isoforms. Sphk isoform 1a has 384 amino acids, isoform 1b has 398 amino acids and isoform 1c has 470 acids. A short stretch of the N-terminus is shown. Identical amino acid residues are indicated in upper-case, while similar amino acid residues are shown in lower-case. (B) RT–PCR analysis of Sphk1 transcripts in HUVEC. Bars a, b and c represent the expression of 384-, 398- and 470-amino-acid isoforms. Results are normalized to GAPDH mRNA levels. Transcripts were not detected in the absence of reverse transcription (results not shown). (C) Immunoblot analysis of the expression of endogenous Sphk1 polypeptides in HUVEC cytosolic fraction is shown. Cytosolic fraction (50 μg) was electrophoresed and probed with rabbit polyclonal anti-Sphk1 antibody.

Article Snippet: Antibody production against Sphk1 and Sphk2 Rabbit polyclonal antisera for GST (glutathione S-transferase)–Sphk1 and GST–Sphk2 fusions were developed by Proteintech Group, and the IgG fraction was purified as described below.

Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction, Expressing, Reverse Transcription, Western Blot

Journal:

Article Title: Extracellular export of sphingosine kinase-1a contributes to the vascular S1P gradient

doi: 10.1042/BJ20060251

Figure Lengend Snippet: HEK-293 cells transiently transfected with pcDNA 3.0 (M), Sphk-1a (1a), Sphk-1b (1b) and Sphk-1c (1c) plasmids were pulsed with [35S]methionine/cysteine. (A) Total cellular extracts (intracellular) and (B) CM (extracellular) were subjected to immunoprecipitation with anti-Sphk1 antibody.

Article Snippet: Antibody production against Sphk1 and Sphk2 Rabbit polyclonal antisera for GST (glutathione S-transferase)–Sphk1 and GST–Sphk2 fusions were developed by Proteintech Group, and the IgG fraction was purified as described below.

Techniques: Transfection, Immunoprecipitation

Journal:

Article Title: Extracellular export of sphingosine kinase-1a contributes to the vascular S1P gradient

doi: 10.1042/BJ20060251

Figure Lengend Snippet: (A) Immunoblot analysis of Sphk1 isoforms in HEK-293 cells. HEK-293 cells were transfected with 3 μg of pcDNA 3.0 (M), hSphk-1a (1a), hSphk-1b (1b) and hSphk-1c (1c) plasmids and 10 μg of the total homogenate was subjected to immunoblot analysis with rabbit polyclonal Sphk1 antibody. (B, C) Phosphorylation of sphingosine by Sphk1 isoforms. Total cellular extract (10 μg) (B) and 0.25 ml of CM (C) from corresponding isoforms were directly assayed for Sphk activity in vitro by addition of sphingosine and [32P]ATP, and the formation of [32P]S1P was analysed by TLC. TLC bands corresponding to [32P]S1P are shown in (B, C). Results shown are the means±S.E.M. (n=3).

Article Snippet: Antibody production against Sphk1 and Sphk2 Rabbit polyclonal antisera for GST (glutathione S-transferase)–Sphk1 and GST–Sphk2 fusions were developed by Proteintech Group, and the IgG fraction was purified as described below.

Techniques: Western Blot, Transfection, Phospho-proteomics, Activity Assay, In Vitro

Journal:

Article Title: Extracellular export of sphingosine kinase-1a contributes to the vascular S1P gradient

doi: 10.1042/BJ20060251

Figure Lengend Snippet: (A) [35S]methionine/cysteine-labelled HUVEC extracts (intracellular) and (B) CM (extracellular) were immunoprecipitated with anti-Sphk1 antibody or NRS for the indicated time and 35S-labelled Sphk1 was quantified by a phosphoimager. Decay of intracellular Sphk-1a is plotted on a semi-logarithmic scale. Results shown are from a representative experiment that was repeated three times.

Article Snippet: Antibody production against Sphk1 and Sphk2 Rabbit polyclonal antisera for GST (glutathione S-transferase)–Sphk1 and GST–Sphk2 fusions were developed by Proteintech Group, and the IgG fraction was purified as described below.

Techniques: Immunoprecipitation

Journal:

Article Title: Extracellular export of sphingosine kinase-1a contributes to the vascular S1P gradient

doi: 10.1042/BJ20060251

Figure Lengend Snippet: Representative confocal images of HEK-293 cells transiently transfected with Mock (pcDNA 3.0), Sphk-1a, Sphk-1b, Sphk-1c, Sphk-2a and Sphk-2b plasmids. Confocal images were collected from fixed cells that were immunostained with polyclonal Sphk1 or Sphk2 antibody. For each construct, the x–y image (centre), x–z (box below, section indicated with horizontal arrowhead) and y–z (right box, section indicated with vertical horizontal arrowhead) images are shown. Note that among the Sphk1 constructs, the b and the c isoforms are more associated with the membrane and the c isoform exhibits a granular localization in the cytoplasm. Sphk-2a is present in the nucleus, whereas the Sphk-2b is not.

Article Snippet: Antibody production against Sphk1 and Sphk2 Rabbit polyclonal antisera for GST (glutathione S-transferase)–Sphk1 and GST–Sphk2 fusions were developed by Proteintech Group, and the IgG fraction was purified as described below.

Techniques: Transfection, Construct, Membrane

Journal:

Article Title: Extracellular export of sphingosine kinase-1a contributes to the vascular S1P gradient

doi: 10.1042/BJ20060251

Figure Lengend Snippet: HUVEC were immunostained with polyclonal Sphk1 and Sphk2 antibodies and visualized in a confocal immunofluorescence microscope as described. Sphk1 isoforms are localized to cytosol, membranous compartments and vesicle-like structures. In contrast, Sphk2 is localized to nuclear and perinuclear region, in addition to cytosol.

Article Snippet: Antibody production against Sphk1 and Sphk2 Rabbit polyclonal antisera for GST (glutathione S-transferase)–Sphk1 and GST–Sphk2 fusions were developed by Proteintech Group, and the IgG fraction was purified as described below.

Techniques: Immunofluorescence, Microscopy

Journal:

Article Title: Extracellular export of sphingosine kinase-1a contributes to the vascular S1P gradient

doi: 10.1042/BJ20060251

Figure Lengend Snippet: Sphk activity in whole blood, platelet-rich and platelet-poor fractions in Sphk1 +/+ and Sphk1 −/− mice A volume of 0.025 ml of whole blood, PRP and PPP fractions was incubated in the presence and absence of 20 μM sphingosine and 10 μCi of [ 32 P]ATP in 500 μM ATP as described in the Materials and methods section. Formation of [ 32 P]S1P was evaluated by TLC analysis. In addition, the formation of [ 32 P]S1P was also evaluated with boiled plasma and in the presence of Sphk inhibitor DMS (50 μM). Results are means±S.E.M. for two independent assays on three to four animals ( n =6–8;). ND, not detected.

Article Snippet: Antibody production against Sphk1 and Sphk2 Rabbit polyclonal antisera for GST (glutathione S-transferase)–Sphk1 and GST–Sphk2 fusions were developed by Proteintech Group, and the IgG fraction was purified as described below.

Techniques: Activity Assay, Incubation, Clinical Proteomics

Journal:

Article Title: Extracellular export of sphingosine kinase-1a contributes to the vascular S1P gradient

doi: 10.1042/BJ20060251

Figure Lengend Snippet: Sphk activity in whole blood and PPP and in PPP–Sphk1 antibody immune complex Human blood, PPP and PPP immunocomplexed with Sphk1 antibody to Protein A beads were assayed in the presence and absence of 20 μM sphingosine and 10 μCi of [ 32 P]ATP in 500 μM ATP as described in the Materials and methods section. Formation of [ 32 P]S1P was evaluated by TLC analysis. PPP immunocomplexed with NRS was used as a control. Formation of [ 32 P]S1P was not detected in the absence of sphingosine. Results are means±S.E.M. ( n =3).

Article Snippet: Antibody production against Sphk1 and Sphk2 Rabbit polyclonal antisera for GST (glutathione S-transferase)–Sphk1 and GST–Sphk2 fusions were developed by Proteintech Group, and the IgG fraction was purified as described below.

Techniques: Activity Assay, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inducible IL-33 expression by mast cells is regulated by a calcium-dependent pathway 1

doi: 10.4049/jimmunol.1201224

Figure Lengend Snippet: BMMCs were transfected with scrambled (top), Sphk1 (middle) or Sphk2 (bottom) shRNA plasmids that also express GFP for 7 days and activated with IgE/DNP. Cells transfected with shRNA plasmids were gated on GFP+ cells. IL-33 protein expression of shRNA transfected cells (GFP+ gated) was analyzed by intracellular staining 24 hours after stimulation. Data is representative of 4 independent experiments.

Article Snippet: APC-anti-rat IgG F(ab′) was from eBioscience. pGFP-V-RS plasmids with or without scrambled, Sphk1 or Sphk2 shRNA were purchased from Origene AMAXA mouse dendritic cell transfection buffer was obtained from Lonza.

Techniques: Transfection, shRNA, Expressing, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Inducible IL-33 expression by mast cells is regulated by a calcium-dependent pathway 1

doi: 10.4049/jimmunol.1201224

Figure Lengend Snippet: IL-33 expression in mast cells upon cross-linking of IgE receptors is regulated by a PI3K-Sphk1-S1P-NFAT pathway. NF-κB is dispensable for the expression of IL-33 and, instead regulates IL-1β. S1P receptor also participates in regulating IL-33 expression.

Article Snippet: APC-anti-rat IgG F(ab′) was from eBioscience. pGFP-V-RS plasmids with or without scrambled, Sphk1 or Sphk2 shRNA were purchased from Origene AMAXA mouse dendritic cell transfection buffer was obtained from Lonza.

Techniques: Expressing

Journal: Molecular Medicine

Article Title: CXCL4 deficiency limits M4 macrophage infiltration and attenuates hyperoxia-induced lung injury

doi: 10.1186/s10020-024-01043-y

Figure Lengend Snippet: M4 macrophages are activated and CXCL4 is upregulated in BPD. ( A ) Representative images of H&E staining (arrow represented the pathological structural change in the lung tissues of mice) and IF for F4/80 (a macrophage marker), S100A8, MMP7, and DAPI in lung tissues from NOX (21% O2) and HYX (95% O2) groups ( n = 6). ( B ) Flow cytometric analysis showing the percentage of F4/80 + S100A8 + MMP7 + cells in NOX and HYX groups ( n = 6). ( C - D ) qRT-PCR analysis and ELISA quantification of CXCL4 expression levels ( n = 6). ( E ) IF staining for F4/80 and CXCL4 in lung tissues ( n = 6). ( F - G ) Western blot analysis of CXCL4, TNF-α, IL-6, MMP7, S100A8, and GAPDH in lung tissues from NOX and HYX groups ( n = 6)

Article Snippet: The primary antibodies used included CXCL4 (ab134087, Abcam, UK), TNF-α (3707, Cell Signaling, USA), IL-6 (12912, Cell Signaling, USA), MMP7 (ab51072, Abcam, UK), S100A8 (MAB4570, R&D Systems, USA), GAPDH (5174, Cell Signaling, USA), SPHK1 (12071, Cell Signaling, USA), SPHK2 (ab107146, Abcam, UK), SPT2 (sc-365859, Santa Cruz Biotechnology, USA), S1PL (PA5-53002, Thermo Fisher, USA), p-SMAD2 (3108, Cell Signaling, USA), and SMAD2 (5339, Cell Signaling, USA), with GAPDH serving as the loading control.

Techniques: Staining, Marker, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot

Journal: Molecular Medicine

Article Title: CXCL4 deficiency limits M4 macrophage infiltration and attenuates hyperoxia-induced lung injury

doi: 10.1186/s10020-024-01043-y

Figure Lengend Snippet: Deletion of CXCL4 prevents the progression of M4 macrophages in the lung. ( A ) IF staining for F4/80, S100A8, and DAPI in lung tissues from WT and CXCL4 KO mice under NOX (21% O 2 ) and HYX (95% O 2 ) conditions ( n = 6). ( B ) IF staining for F4/80, MMP7, and DAPI ( n = 6). ( C ) Flow cytometry analysis quantifying F4/80 + S100A8 + MMP7 + cells (%) ( n = 6). ( D - E ) Western blot analysis of CXCL4, TNF-α, IL-6, MMP7, S100A8, and GAPDH ( n = 6)

Article Snippet: The primary antibodies used included CXCL4 (ab134087, Abcam, UK), TNF-α (3707, Cell Signaling, USA), IL-6 (12912, Cell Signaling, USA), MMP7 (ab51072, Abcam, UK), S100A8 (MAB4570, R&D Systems, USA), GAPDH (5174, Cell Signaling, USA), SPHK1 (12071, Cell Signaling, USA), SPHK2 (ab107146, Abcam, UK), SPT2 (sc-365859, Santa Cruz Biotechnology, USA), S1PL (PA5-53002, Thermo Fisher, USA), p-SMAD2 (3108, Cell Signaling, USA), and SMAD2 (5339, Cell Signaling, USA), with GAPDH serving as the loading control.

Techniques: Staining, Flow Cytometry, Western Blot

Journal: American Journal of Translational Research

Article Title: Reduction of COX-2 through modulating miR-124/SPHK1 axis contributes to the antimetastatic effect of alpinumisoflavone in melanoma

doi:

Figure Lengend Snippet: AIF represses COX-2 expression by modulating miR-124/SPHK1 signaling. Cells were pretreated for 24 hours with AIF (10 μM unless otherwise noted). A. AIF treatment dose-dependently decreased SPHK1 protein levels in melanoma cells. B. AIF treatment dose-dependently increased mIR-124 expression in melanoma cells. C. MiR-124 knockdown significantly attenuated the suppressive effects of AIF on SPHK1 expression, as demonstrated by western blot analysis. D. Overexpression of miR-124 significantly decreased SPHK1 transcription, as assessed by the luciferase reporter assay. E. Both MiR-124 knockdown and SPHK1 overexpression significantly attenuated the suppressive effect of AIF on COX-2 mRNA expression. F. Both MiR-124 knockdown and SPHK1 overexpression significantly attenuated the suppressive effect of AIF on COX-2 protein expression. **P < 0.01 vs. control, ^^P < 0.01 vs. AIF.

Article Snippet: Silencing COX-2 or SPHK1 in melanoma cells The siRNA oligos for COX-2 or SPHK1 gene knockdown were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Expressing, Knockdown, Western Blot, Over Expression, Luciferase, Reporter Assay, Control

Journal: American Journal of Translational Research

Article Title: Reduction of COX-2 through modulating miR-124/SPHK1 axis contributes to the antimetastatic effect of alpinumisoflavone in melanoma

doi:

Figure Lengend Snippet: MiR-124/SPHK1 signaling is involved in the antimetastatic effect of AIF. Cells were pretreated for 24 hours with AIF (10 μM if not otherwise noted). A. COX-2 overexpression attenuated the effect of AIF on melanin content in melanoma cells. B. Both MiR-124 knockdown and SPHK1 overexpression significantly attenuated the effect of AIF on PpIX accumulation. C. Both MiR-124 knockdown and SPHK1 overexpression significantly attenuated the effect of AIF on TG2 activity. D. Both MiR-124 knockdown and SPHK1 overexpression significantly attenuated AIF-mediated inhibition of cell migration. E. Both MiR-124 knockdown and SPHK1 overexpression significantly attenuated AIF-mediated inhibition of cell invasion. **P < 0.01.

Article Snippet: Silencing COX-2 or SPHK1 in melanoma cells The siRNA oligos for COX-2 or SPHK1 gene knockdown were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Over Expression, Knockdown, Activity Assay, Inhibition, Migration

Journal: American Journal of Translational Research

Article Title: Reduction of COX-2 through modulating miR-124/SPHK1 axis contributes to the antimetastatic effect of alpinumisoflavone in melanoma

doi:

Figure Lengend Snippet: AIF suppresses B16-F10 murine melanoma cell lung metastasis in vivo. Xenograft-bearing mice were treated for 24 days with vehicle or AIF (20 or 50 mg/kg/day) by intragastric administration. Representative images of lung metastasis and the number of metastatic nodules in the lungs are shown. A. AIF significantly suppressed lung metastasis in vivo. B. AIF increased miR-124 expression in vivo. C. AIF treatment decreased expression levels of COX-2 and SPHK1 in vivo. *P < 0.05 (n = 8).

Article Snippet: Silencing COX-2 or SPHK1 in melanoma cells The siRNA oligos for COX-2 or SPHK1 gene knockdown were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: In Vivo, Expressing