Journal: Journal of Inflammation (London, England)

Article Title: Enhanced inflammasome activation and reduced sphingosine-1 phosphate S1P signalling in a respiratory mucoobstructive disease model

doi: 10.1186/s12950-020-00248-2

Figure Lengend Snippet: Colocalization and correlation of SPHK1 expression with SPNS2. a Representative confocal images of a control animal bronchiole showing colocalization of SPHK1 (red) and SPNS2 (green). Yellow is merged color of red and green. Blue is DAPI. Scale bars are in micrometers. b Positive Spearman’s correlation between MFI of SPHK1 and SPNS2 in both unobstructed (red; r = 0.640; p < 0.001; n = 62 bronchioles from 7 mice) and mucus obstructed (green; r = 0.745; p < 0.001; n = 42 bronchioles from 4 mice)

Article Snippet: Primary antibodies included rabbit polyclonal to SPHK1 and SPHK2 (Bs-2652R and Bs-2653R, Bioss, Woburn, MA, USA, both 1:50), NLRP3 and IL-1β (sc-66,846 and sc-7884, Santa Cruz, Dallas, TX, USA, both 1:40), GLUT1 (Glucose Transporter 1, ab652, Abcam, Cambridge, UK, 1:100), goat polyclonal antibody to SPNS2 (sc-165,572, Santa Cruz, 1:40), TNFα (sc-1348, Santa Cruz, 1:50), myeloperoxidase (#2141, Osenses, Adelaide, SA, Australia, 1:100), and a mouse monoclonal antibody to neutrophil elastase (M0752, Dako GmbH, Jena, Germany, 1:100).

Techniques: Expressing

Journal: Journal of Inflammation (London, England)

Article Title: Enhanced inflammasome activation and reduced sphingosine-1 phosphate S1P signalling in a respiratory mucoobstructive disease model

doi: 10.1186/s12950-020-00248-2

Figure Lengend Snippet: Hypothetical model of NLRP3 inflammasome activation and S1P signalling dysregulation in mucus-obstructed bronchioles. Unobstructed bronchioles (AIR): NLRP3 is mostly in monomeric state, only background levels of cleaved IL-1β are detected; SPHK1 generates cytosolic S1P, part of which is exported via SPNS2 for extracellular (inside-out) signalling via S1PRs; SPHK2 generates S1P mostly for intracellular signalling. Obstructed bronchioles (MUCUS): Increased oligomerization of NLRP3 (inflammasome activation) in either epithelium or infiltrating leucocytes results in IL-1β cleavage and increased luminal specks of NLRP3/IL-1β; decrease of SPNS2 and SPHK2 results in reduction of extra- and intracellular S1P signalling

Article Snippet: Primary antibodies included rabbit polyclonal to SPHK1 and SPHK2 (Bs-2652R and Bs-2653R, Bioss, Woburn, MA, USA, both 1:50), NLRP3 and IL-1β (sc-66,846 and sc-7884, Santa Cruz, Dallas, TX, USA, both 1:40), GLUT1 (Glucose Transporter 1, ab652, Abcam, Cambridge, UK, 1:100), goat polyclonal antibody to SPNS2 (sc-165,572, Santa Cruz, 1:40), TNFα (sc-1348, Santa Cruz, 1:50), myeloperoxidase (#2141, Osenses, Adelaide, SA, Australia, 1:100), and a mouse monoclonal antibody to neutrophil elastase (M0752, Dako GmbH, Jena, Germany, 1:100).

Techniques: Activation Assay

Journal: Scientific reports

Article Title: Clinical relevance of CERK and SPHK1 in breast cancer and their association with metastasis and drug resistance.

doi: 10.1038/s41598-022-20976-0

Figure Lengend Snippet: Figure 3. Correlation of SPHK1 gene expression with various clinicopathological characteristics in breast tumor tissues. (A) pN Stage (pN0 + pNx- [Nodal Negative], pN1 + pN2-[Nodal Positive]) (B) pTNM Stage (pTNM I + II- [early stage], pTNM III + IV- [late stage]) (C) Correlation with Ki67.

Article Snippet: Primary antibodies against SPHK1 (NBP2-20472); CERK (NB1002911); ABCC1 (NB400-156); ABCG2 (NBP2-22124); MMP-2 (NBP2-27208SS); MMP-9 (NBP2-13173SS) were purchased from Novus Biologicals (Centennial, Colorado, United States).

Techniques: Gene Expression

Journal: Scientific reports

Article Title: Clinical relevance of CERK and SPHK1 in breast cancer and their association with metastasis and drug resistance.

doi: 10.1038/s41598-022-20976-0

Figure Lengend Snippet: Figure 4. Protein expression of SPHK1 and CERK in breast cancer patients. (A) Representative blots in adjacent normal (N) and tumor (T) tissues, (B) Densitometric analysis of SPHK1 and (C) CERK levels in adjacent normal and tumor tissues.

Article Snippet: Primary antibodies against SPHK1 (NBP2-20472); CERK (NB1002911); ABCC1 (NB400-156); ABCG2 (NBP2-22124); MMP-2 (NBP2-27208SS); MMP-9 (NBP2-13173SS) were purchased from Novus Biologicals (Centennial, Colorado, United States).

Techniques: Expressing

Journal: Scientific reports

Article Title: Clinical relevance of CERK and SPHK1 in breast cancer and their association with metastasis and drug resistance.

doi: 10.1038/s41598-022-20976-0

Figure Lengend Snippet: Figure 6. Network-based analysis of interaction network corresponding to candidate genes. (A) The node degree distribution of PPI network. The number of genes is plotted as a function of their degree reflecting a power-law like distribution. The red line corresponds to a power-law distribution. (B) Sub-network of candidate genes. The large-sized black-colored nodes represent the candidate genes (ABCC1, ABCG2, CERK, MMP-2, MMP-9, and SPHK1), while the small gray color nodes represent the corresponding interactor genes. Interactions are represented in gray color and the depth of color represents the strength of correlations. (C) Shortest path lengths among candidate genes. Heatmap of shortest path length among the candidate genes, where the values represent the number of shortest paths between any pair.

Article Snippet: Primary antibodies against SPHK1 (NBP2-20472); CERK (NB1002911); ABCC1 (NB400-156); ABCG2 (NBP2-22124); MMP-2 (NBP2-27208SS); MMP-9 (NBP2-13173SS) were purchased from Novus Biologicals (Centennial, Colorado, United States).

Techniques:

Journal: Scientific reports

Article Title: Clinical relevance of CERK and SPHK1 in breast cancer and their association with metastasis and drug resistance.

doi: 10.1038/s41598-022-20976-0

Figure Lengend Snippet: Figure 8. Correlation of CERK with (A) MMP-2, (B) MMP-9 and SPHK1 with (C) MMP-2, (D) MMP-9 in local cohort and CERK with (E) MMP-2, (F) MMP-9 and SPHK1 with (G) MMP-2, (H) MMP-9 in TCGA cohort.

Article Snippet: Primary antibodies against SPHK1 (NBP2-20472); CERK (NB1002911); ABCC1 (NB400-156); ABCG2 (NBP2-22124); MMP-2 (NBP2-27208SS); MMP-9 (NBP2-13173SS) were purchased from Novus Biologicals (Centennial, Colorado, United States).

Techniques:

Journal: Scientific reports

Article Title: Clinical relevance of CERK and SPHK1 in breast cancer and their association with metastasis and drug resistance.

doi: 10.1038/s41598-022-20976-0

Figure Lengend Snippet: Figure 10. Correlation of CERK with (A) ABCC1, (B) ABCG2 and SPHK1 with (C) ABCC1, (D) ABCG2 in local cohort and CERK with (E) ABCC1, (F) ABCG2 and SPHK1 with (G) ABCC1, (H) ABCG2 in TCGA cohort.

Article Snippet: Primary antibodies against SPHK1 (NBP2-20472); CERK (NB1002911); ABCC1 (NB400-156); ABCG2 (NBP2-22124); MMP-2 (NBP2-27208SS); MMP-9 (NBP2-13173SS) were purchased from Novus Biologicals (Centennial, Colorado, United States).

Techniques:

Journal: Scientific reports

Article Title: Clinical relevance of CERK and SPHK1 in breast cancer and their association with metastasis and drug resistance.

doi: 10.1038/s41598-022-20976-0

Figure Lengend Snippet: Figure 11. Expression of SPNS2 in TCGA cohort. (A) Relative gene expression between adjacent normal and tumor tissue, (B) Correlation with SPHK1.

Article Snippet: Primary antibodies against SPHK1 (NBP2-20472); CERK (NB1002911); ABCC1 (NB400-156); ABCG2 (NBP2-22124); MMP-2 (NBP2-27208SS); MMP-9 (NBP2-13173SS) were purchased from Novus Biologicals (Centennial, Colorado, United States).

Techniques: Expressing, Gene Expression

Journal: Molecular Medicine Reports

Article Title: Ginsenoside Rg1 exerts anti-apoptotic effects on non-alcoholic fatty liver cells by downregulating the expression of SGPL1

doi: 10.3892/mmr.2022.12694

Figure Lengend Snippet: Effect of Rg1 on sphingolipid pathway proteins. (A) SPHK1, SGPL1, p-Akt, p-Erk1/2, Akt and Erk1/2 expression levels were detected in HHL-5 cells treated with Rg1 (0.2, 0.4 and 0.6 mM) after exposure to MCE (1%) by western blotting. (B) SPHK1 and SGPL1 mRNA expression levels were detected in HHL-5 cells using reverse transcription-quantitative PCR. (C) SPHK1 and SGPL1 protein expression levels were normalized to actin expression, and p-Akt and p-Erk1/2 protein expression levels were normalized to Akt or Erk1/2 using ImageJ software. *P<0.05, **P<0.01 and ***P<0.001; MCE, medium- and long-chain fat emulsion; Mod, model; p-, phosphorylated; Rec, recovery; SGPL1, sphingosine-1-phosphate lyase 1; SPHK1, sphingosine kinase 1; Rg1, ginsenoside Rg1.

Article Snippet: The antibody against SPHK1 (AF5536) was obtained from R&D Systems. β-actin antibody (1:8,000; cat. no. A1978; MilliporeSigma) was used as a loading control.

Techniques: Expressing, Western Blot, Reverse Transcription, Real-time Polymerase Chain Reaction, Software, Emulsion

Journal: Molecular Medicine Reports

Article Title: Ginsenoside Rg1 exerts anti-apoptotic effects on non-alcoholic fatty liver cells by downregulating the expression of SGPL1

doi: 10.3892/mmr.2022.12694

Figure Lengend Snippet: Molecular mechanism underlying the inhibitory effects of Rg1 on apoptosis in steatotic HHL-5 hepatocytes. MCE, medium- and long-chain fat emulsion; SPHK1, sphingosine kinase 1; SGPL1, sphingosine-1-phosphate lyase 1; S1P, sphingosine-1-phosphate; Mt, mitochondria; Rg1, ginsenoside Rg1.

Article Snippet: The antibody against SPHK1 (AF5536) was obtained from R&D Systems. β-actin antibody (1:8,000; cat. no. A1978; MilliporeSigma) was used as a loading control.

Techniques: Emulsion

Journal: Scientific reports

Article Title: Peretinoin, an acyclic retinoid, inhibits hepatocarcinogenesis by suppressing sphingosine kinase 1 expression in vitro and in vivo.

doi: 10.1038/s41598-017-17285-2

Figure Lengend Snippet: Figure 1. Liver mRNA levels of SPHK1 in the PDGFC-Tg and Ath-HF diet mouse models. (a) mRNA level of SPHK1 in the liver of PDGF-C Tg mice determined by microarray analysis. We normalized the mRNA level of SPHK1 in the liver of PDGF-C Tg mice treated with or without 0.06% peretinoin to that of non-Tg mice. The mRNA levels were calculated from the microarray data published in one of our previous reports16. This figure shows the relative mRNA level of SPHK1 under each condition to that of non-Tg mice. Error bars indicate the standard deviation from three mice. The statistical significance of the difference in the average between the two groups was analyzed by the Student’s t test. (b) mRNA levels in the liver in the Ath-HF diet mouse model determined by microarray analysis. We normalized the mRNA level of SPHK1 in the liver of Ath-HF diet mice with or without 0.03% peretinoin to that of LF mice. The mRNA levels were calculated from the microarray data published in one of our previous reports17. This figure shows the relative mRNA level of SPHK1 under each condition to that in LF mice. Error bars indicate the standard deviation from three mice. The statistical significance was analyzed as above. (c) mRNA level of the liver in the Ath-HF diet mouse model determined by qRT-PCR. We quantitated the mRNA levels of SPHK1 and β-actin in the liver of Ath-HF diet mice with or without 0.03% peretinoin to that of LF mice by qRT-PCR (TaqMan assay); the mRNA level of SPHK1 was then normalized to that of β-actin for each mouse. The normalized mRNA levels of SPHK1 in the liver of Ath-HF diet mice with or without peretinoin were further normalized to that of LF mice. This figure shows the relative mRNA level of SPHK1 of each condition to that of LF mice. Error bars indicate the standard deviation from at least 10 mice. Statistical significance was analyzed as above. *p < 0.05, ***p < 0.005.

Article Snippet: The membranes were blocked in Blocking One or Blocking One-P solution (Nacalai Tesque, Kyoto, Japan), and the expression of SPHK1 was evaluated with rabbit anti-SPHK1 (#A302-177A; Bethyl Laboratories, Montgomery, TX), rabbit anti-Sp1 (#5931; Cell Signaling Technology, Danvers, MA), and rabbit β-actin antibody (#4967; Cell Signaling Technology).

Techniques: Microarray, Standard Deviation, Quantitative RT-PCR, TaqMan Assay

Journal: Scientific reports

Article Title: Peretinoin, an acyclic retinoid, inhibits hepatocarcinogenesis by suppressing sphingosine kinase 1 expression in vitro and in vivo.

doi: 10.1038/s41598-017-17285-2

Figure Lengend Snippet: Figure 2. mRNA level of SPHK1 in human liver. A. comparison of the mRNA level of SPHK1 between mild and severe fibrosis in the HCV-infected liver. (a) We performed liver biopsies on patients infected with HCV prior to antiviral treatment and quantified the mRNA levels of SPHK1 and β-actin in total RNA extracted from the liver by qRT-PCR (TaqMan assay); the mRNA level of SPHK1 was then normalized to that of β-actin for each patient. The patients were categorized into two groups based on their liver fibrosis grade; mild fibrosis was defined as METAVIR Score F1 or F2 and severe fibrosis as F3 or F4. We compared the normalized mRNA level of SPHK1 between mild and severe fibrotic liver. Error bars show the standard deviation from 119 patients with mild liver fibrosis and 59 patients with severe fibrosis. The statistical significance of the difference in the average between the two groups was analyzed by the Student’s t test. (b) Comparison of the mRNA level of SPHK1 before and after HCV eradication. We performed liver biopsies on another 12 patients before they started antiviral treatment for HCV and after successful eradication of HCV. We quantitated the mRNA levels of SPHK1 and β-actin in total RNA extracted from the liver by qRT-PCR (TaqMan assay); the mRNA level of SPHK1 was then normalized to that of β-actin for each patient. The relative mRNA level of SPHK1 after HCV eradication (post-SVR [sustained virologic response]) was normalized to that before antiviral treatment (prior to antiviral treatment) in the individual patients, with the mRNA level of SPHK1 before antiviral treatment set to 1. The statistical significance of the difference in the average between these two groups was analyzed by a paired t test. *p < 0.05, ***p < 0.005.

Article Snippet: The membranes were blocked in Blocking One or Blocking One-P solution (Nacalai Tesque, Kyoto, Japan), and the expression of SPHK1 was evaluated with rabbit anti-SPHK1 (#A302-177A; Bethyl Laboratories, Montgomery, TX), rabbit anti-Sp1 (#5931; Cell Signaling Technology, Danvers, MA), and rabbit β-actin antibody (#4967; Cell Signaling Technology).

Techniques: Comparison, Infection, Quantitative RT-PCR, TaqMan Assay, Standard Deviation

Journal: Scientific reports

Article Title: Peretinoin, an acyclic retinoid, inhibits hepatocarcinogenesis by suppressing sphingosine kinase 1 expression in vitro and in vivo.

doi: 10.1038/s41598-017-17285-2

Figure Lengend Snippet: Figure 3. Effects of peretinoin on the mRNA level, protein expression, and enzymatic activity of SPHK1. (a) effects of peretinoin on the mRNA level of sphingolipid-related genes in Huh-7 cells. Huh-7 cells were treated with peretinoin at 10, 25, or 50 μM or with 0.5% DMSO for 72 h and total cellular RNA was extracted. The mRNA levels of SPHK1, SPHK2, S1P lyase, S1P receptor 1–3 (S1P1, S1P2, and S1P3), and β-actin were quantitated by qRT-PCR (SYBR green assay), and the mRNA levels of SPHK1, SPHK2, S1P lyase, S1P1, S1P2, and S1P3 were normalized to that of β-actin. The relative mRNA level of each gene under each condition was normalized to that of DMSO control. (b) Effects of peretinoin on the protein expression of SPHK1 in Huh-7 cells. Huh-7 cells were treated with 0.5% DMSO or peretinoin 10, 20, or 40 μM. Total cell lysates were collected 12, 24, 48, and 72 h later and then probed by western blotting with anti-SPHK1 and β-actin antibodies. Full- length gels and blots before cropping are shown in supplemental Fig. S8. (c) Effects of peretinoin on the enzymatic activity of SPHK1 in Huh-7 cells. Huh-7 cells were treated with 0.5% DMSO or peretinoin at 10, 20, and 40 μM and a plasmid encoding SPHK1 cDNA or an empty vector were transfected into Huh-7 cells.

Article Snippet: The membranes were blocked in Blocking One or Blocking One-P solution (Nacalai Tesque, Kyoto, Japan), and the expression of SPHK1 was evaluated with rabbit anti-SPHK1 (#A302-177A; Bethyl Laboratories, Montgomery, TX), rabbit anti-Sp1 (#5931; Cell Signaling Technology, Danvers, MA), and rabbit β-actin antibody (#4967; Cell Signaling Technology).

Techniques: Expressing, Activity Assay, Quantitative RT-PCR, SYBR Green Assay, Control, Western Blot, Plasmid Preparation, Transfection

Journal: Scientific reports

Article Title: Peretinoin, an acyclic retinoid, inhibits hepatocarcinogenesis by suppressing sphingosine kinase 1 expression in vitro and in vivo.

doi: 10.1038/s41598-017-17285-2

Figure Lengend Snippet: Figure 4. Effects of peretinoin on SPHK1 promoter activity. A effects of peretinoin on the promoter activity of SPHK1 and SPHK2. (a) Plasmids encoding luciferase under the control of the promoter region of SPHK1, SPHK2, β-actin, and GAPDH were transfected into Huh-7 cells and, 24 h later, DMSO or peretinoin at 1, 5, 10, 20, or 40 μM was added. After 48-h DMSO or peretinoin treatment, luciferase activity was determined. The luciferase activity in each condition was normalized to that of DMSO control. Error bars indicate the standard deviation from three experiments and the statistical significance of the difference in the average was analyzed by one-way ANOVA. (b) Possible binding sites of Sp1 in the SPHK1 promoter region and a representation of deletion mutants. This figure shows the possible binding sites of Sp1 in the promoter region of SPHK1 and the series of deletion mutants analyzed. (c) Enhancement of the promoter activity of SPHK1 by Sp1 overexpression. A plasmid encoding Sp1 or a control empty vector was co-transfected with a plasmid encoding luciferase under the control of the SPHK1 promoter region and, 48 h later, luciferase activity was determined. Error bars indicate the standard deviation from three experiments and the statistical significance of the difference in the average was analyzed by the Student’s t test. (d) Restoration of the suppressive effects of peretinoin on the

Article Snippet: The membranes were blocked in Blocking One or Blocking One-P solution (Nacalai Tesque, Kyoto, Japan), and the expression of SPHK1 was evaluated with rabbit anti-SPHK1 (#A302-177A; Bethyl Laboratories, Montgomery, TX), rabbit anti-Sp1 (#5931; Cell Signaling Technology, Danvers, MA), and rabbit β-actin antibody (#4967; Cell Signaling Technology).

Techniques: Activity Assay, Luciferase, Control, Transfection, Standard Deviation, Binding Assay, Over Expression, Plasmid Preparation

Journal: Scientific reports

Article Title: Peretinoin, an acyclic retinoid, inhibits hepatocarcinogenesis by suppressing sphingosine kinase 1 expression in vitro and in vivo.

doi: 10.1038/s41598-017-17285-2

Figure Lengend Snippet: Figure 5. Effects of SPHK1 knockout on hepatocarcinogenesis in a DEN-induced hepatoma mouse model. DEN was injected into the peritoneal cavity of 2-week-old SPHK1 knockout, SPHK1 Tg, and wild-type mice at 25 mg/kg, and then those mice were sacrificed at 40 weeks old. (a) The numbers of mice that had a liver tumor at sacrifice. (b) The numbers of liver tumors in each mouse. (c) The maximum liver tumor size in individual mice that developed hepatoma. (d) Macrophotographs of representative mouse livers at sacrifice. (e) High power field microphotographs of representative mouse liver tumors stained with hematoxylin and eosin. Error bars shows the standard deviation from at least 20 mice, and the statistical significance of the difference in the average between these two groups was analyzed by the Student’s t test. *p < 0.05, ***p < 0.005

Article Snippet: The membranes were blocked in Blocking One or Blocking One-P solution (Nacalai Tesque, Kyoto, Japan), and the expression of SPHK1 was evaluated with rabbit anti-SPHK1 (#A302-177A; Bethyl Laboratories, Montgomery, TX), rabbit anti-Sp1 (#5931; Cell Signaling Technology, Danvers, MA), and rabbit β-actin antibody (#4967; Cell Signaling Technology).

Techniques: Knock-Out, Injection, Staining, Standard Deviation

Journal: European Journal of Histochemistry : EJH

Article Title: Interaction between sphingosine kinase/sphingosine 1 phosphate and transforming growth factor-β/Smads pathways in experimental intestinal fibrosis. An in vivo immunohistochemical study

doi: 10.4081/ejh.2018.2956

Figure Lengend Snippet: Antibodies used with their sources and dilutions together with the positive controls.

Article Snippet: SPHK1 , (PA1996) BosterBio, Pleasanton, CA, USA , 1:100 , Human kidney cancer.

Techniques:

Journal: European Journal of Histochemistry : EJH

Article Title: Interaction between sphingosine kinase/sphingosine 1 phosphate and transforming growth factor-β/Smads pathways in experimental intestinal fibrosis. An in vivo immunohistochemical study

doi: 10.4081/ejh.2018.2956

Figure Lengend Snippet: Immunohistochemistry (original magnification 10x). The immunopositivity of SPHK1, RhoA and PI3K is prevalent in DSS mice (B,E,H) respect to control (A,D,G). In particular, in DSS mice, SPHK1 expression is predominantly localized in mucosa layer and in submucosa of colon (B). In the same group RhoA and PI3K staining results highly expressed (E,H) in mucosa, especially in the ephitelium (supplementary Figure 1), but also in submucosa and in serosa.

Article Snippet: SPHK1 , (PA1996) BosterBio, Pleasanton, CA, USA , 1:100 , Human kidney cancer.

Techniques: Immunohistochemistry, Control, Expressing, Staining

Journal: European Journal of Histochemistry : EJH

Article Title: Interaction between sphingosine kinase/sphingosine 1 phosphate and transforming growth factor-β/Smads pathways in experimental intestinal fibrosis. An in vivo immunohistochemical study

doi: 10.4081/ejh.2018.2956

Figure Lengend Snippet: Schematic representation of TGF-β dependent signaling implicated in fibrosis process. TGF-β upon biding its receptors, activates both canonical Smads pathway and noncanonical pathway (SPHK1/S1P/mTOR), inducing transcription of pro-fibrotic target genes.

Article Snippet: SPHK1 , (PA1996) BosterBio, Pleasanton, CA, USA , 1:100 , Human kidney cancer.

Techniques: