Journal: Human Molecular Genetics

Article Title: Inhibiting sphingosine kinase 2 mitigates mutant Huntingtin-induced neurodegeneration in neuron models of Huntington disease

doi: 10.1093/hmg/ddx046

Figure Lengend Snippet: SK2 is nuclear in primary cultured cortical and striatal neurons. (A) Primary cortical and striatal cultures at 14 days in vitro (DIV) were fixed and stained with antibodies against SK2 and MAP2c, and with the nuclear Hoechst dye (DAPI). Scale bar is 30 μm. (B) Primary cortical and striatal cultures at 14 DIV were fixed and stained with antibodies against SK1 and MAP2c, and with DAPI. Scale bar is 30 μm.

Article Snippet: Antibodies against SK1 were from Novus Biologicals (#NBP1-22974; 1:100).

Techniques: Cell Culture, In Vitro, Staining

Journal: Human Molecular Genetics

Article Title: Inhibiting sphingosine kinase 2 mitigates mutant Huntingtin-induced neurodegeneration in neuron models of Huntington disease

doi: 10.1093/hmg/ddx046

Figure Lengend Snippet: Targeting ectopically expressed SK2 to the neuronal nucleus. (A) Previous studies used the SK2 constructs tagged with a fluorescent protein to SK2's C-terminus. Primary cortical neurons were transfected with SK2-GFP, fixed, and stained with an antibody against MAP2c and with DAPI. Scale bar is 10 μm. Arrow depicts a transfected neuron. Transfected neurons exhibit a significant cytosolic localization of the SK2-GFP construct, unlike the endogenous SK2. (B) To target SK2 specifically to the neuronal nucleus, SK2 was fused to mApple (a red fluorescent protein), which contains the SV40 nuclear localization signal (NLS) on its C-terminus. Primary cortical neurons were transfected with SK2-mApple-NLS, fixed, and stained with an antibody against MAP2c and with DAPI. Neurons transfected with SK2-mApple-NLS exhibit nuclear localization of the construct. Scale bar is 10 μm. Arrow depicts a transfected neuron. (C) Primary cortical neurons were nucleofected either with mApple (cont) or SK2-mApple-NLS, plated, and maintained for 3 days. Neuronal lysates were then analyzed by western blotting with antibodies against pan-SK2 (SK2), pan-SK1 (SK1) and phosphorylated SK1 (pSK1). Actin was used as a loading control. (D) Quantification of SK1 and pSK1 band intensities from (C) normalized to actin. n.s., not significant; P = 0.79 and P = 0.37 (t-test) for pSK1 and SK1, respectively. Results were pooled from three independent experiments. (E) Primary cortical neurons were nucleofected with mApple (cont) or SK2-mApple-NLS plated and maintained for 3 days. The levels of S1P were measured by liquid chromatography and mass spectrometry from the nuclear neuronal fraction. ***P = 0.0012 (t-test). Results were pooled from two independent experiments with triplicates in each one.

Article Snippet: Antibodies against SK1 were from Novus Biologicals (#NBP1-22974; 1:100).

Techniques: Construct, Transfection, Staining, Western Blot, Control, Liquid Chromatography, Mass Spectrometry

Journal: Oncotarget

Article Title: Regulation of macrophage migration in ischemic mouse hearts via an AKT2/NBA1/SPK1 pathway

doi: 10.18632/oncotarget.23263

Figure Lengend Snippet: ANA-1 cells were starved for 24 h and were stimulated with 100 ng/ml LPS for 2 h. Immunoblots exhibit total SPK1 (A) , P-SPK1 (B) , SPK2 (C) , P-SPK2 (D) and GAPDH protein expression levels. Graph shows GAPDH normalized SPK1 (A), P-SPK1 (B), SPK2 (C), and P-SPK2 (D) levels. Data are presented as the mean ± SEM; n=3. * P < 0.05, ** P < 0.01 compared with Con; NS=not significant.

Article Snippet: P-SPK1 (catalog number AP 05255PU-N) was from DBA Acris Antibodies (Rockville, MD, USA).

Techniques: Western Blot, Expressing

Journal: Oncotarget

Article Title: Regulation of macrophage migration in ischemic mouse hearts via an AKT2/NBA1/SPK1 pathway

doi: 10.18632/oncotarget.23263

Figure Lengend Snippet: (A) Migration of peritoneal macrophages from WT and AKT2−/− mice. Primary cells enriched for macrophages were placed into 6-well plates (1×10 6 cells/ml) in DMEM plus 10% FBS. After 24 h incubation and starved overnight, cells were plated in the upper well and serum-free RPMI 1640 medium containing 100 ng/ml LPS was added to the bottom well. After 16 h of incubation, cells migrating across the membrane were stained and counted. Four random fields were counted and the number of migrated cells was used as an index for migration. (B-F) Protein expression in LPS induced murine peritoneal macrophages. Whole cell lysates were prepared after cells were stimulated with LPS. Immunoblots show total AKT2 (B), P-AKT2 (C), NBA1 (D), SPK1 (E), P-SPK1 (F), and GAPDH protein expression levels. Graph shows GAPDH normalized AKT2 (B), P-AKT2 (C), NBA1 (D), SPK1 (E), and P-SPK1 (F) levels. Data are presented as the mean ± SEM; n=3. * P < 0.05, ** P < 0.01 compared with WT without LPS; # P < 0.05, ## P < 0.01 compared with WT+LPS induced group; § P<0.05, §§ P<0.01 vs SPK1−/− without ATV treatment group; NS=not significant.

Article Snippet: P-SPK1 (catalog number AP 05255PU-N) was from DBA Acris Antibodies (Rockville, MD, USA).

Techniques: Migration, Incubation, Staining, Expressing, Western Blot

Journal: Oncotarget

Article Title: Regulation of macrophage migration in ischemic mouse hearts via an AKT2/NBA1/SPK1 pathway

doi: 10.18632/oncotarget.23263

Figure Lengend Snippet: First, 5×10 4 ANA-1 cells were infected by NBA1-siRNA infection solution (5×10 8 TU) for 72h, starved overnight and then stimulated with 100 ng/ml LPS for 2h. Whole cell lysates were prepared after cells were stimulated with LPS. Immunoblots show total AKT2 (A) , P-AKT2 (B) , NBA1 (C) , SPK1 (D) , P-SPK1 (E) and GAPDH protein expression levels. Graph shows GAPDH normalized AKT2 (A), P-AKT2 (B), NBA1 (C), SPK1 (D), and P-SPK1 (E) levels. Data are presented as the mean ± SEM; n=3. * P<0.05, ** P < 0.01 compared with Con without LPS; # P<0.05, ## P < 0.01 compared Con+LPS group; NS=not significant.

Article Snippet: P-SPK1 (catalog number AP 05255PU-N) was from DBA Acris Antibodies (Rockville, MD, USA).

Techniques: Infection, Western Blot, Expressing

Journal: Oncotarget

Article Title: Regulation of macrophage migration in ischemic mouse hearts via an AKT2/NBA1/SPK1 pathway

doi: 10.18632/oncotarget.23263

Figure Lengend Snippet: (A) SPK1-siRNA decreased migration of ANA-1 cells in a trans-well assay after LPS induction. ANA-1 cells were infected by LV-SPK1-siRNA, then cells were detached and cell suspension (5×10 5 cells/ml) was placed in the upper well and serum-free RPMI 1640 medium with 100 ng/ml LPS was added to the bottom well. After 16 h of incubation, cells migrating across the membrane were stained and counted. The experiment was repeated at least three times with similar results. (B-G) Protein expression in LPS and SPK1-siRNA induced ANA-1 cells. Whole cell lysates were prepared following stimulation with LPS and SPK1-siRAN. Immunoblots showed P-AKT2 (B), NBA1 (C), SPK1 (D), P-SPK1 (E), SPK2 (F), P-SPK2 (G) and GAPDH protein expression levels. Graph shows GAPDH normalized levels of P-AKT2 (B), NBA1 (C), SPK1 (D), P-SPK1 (E), SPK2 (F), and P-SPK2 (G). Data are presented as the mean ± SEM; n=3. * P < 0.05, ** P < 0.01 compared with Con without LPS; # P < 0.05, ## P < 0.01 compared with Con + LPS induced group; NS=not significant.

Article Snippet: P-SPK1 (catalog number AP 05255PU-N) was from DBA Acris Antibodies (Rockville, MD, USA).

Techniques: Migration, Infection, Incubation, Staining, Expressing, Western Blot

Journal: Oncotarget

Article Title: Regulation of macrophage migration in ischemic mouse hearts via an AKT2/NBA1/SPK1 pathway

doi: 10.18632/oncotarget.23263

Figure Lengend Snippet: (A) ATV decreases macrophage migration. ANA-1 cells were grown overnight and starved for 24 h and detached. Then, 5×10 5 cells were plated in the upper well and serum-free RPMI 1640 medium containing 100 ng/ml LPS with or without 10 μM ATV were added to the bottom well. Cells migrating across the membrane were stained and counted. The experiment was repeated at least three times with similar results. (B-F) ATV decreases AKT2, P-AKT2, NBA1, SPK1, and P-SPK1 protein expressions in LPS induced macrophages. ANA-1 cells were incubated with ATV (10 μM) for 24h, then 100 ng/ml LPS induced ANA-1 cells for 2h. Then whole cell lysates were prepared. Immunoblots show AKT2 (B), P-AKT2 (C), NBA1 (D), SPK1 (E), P-SPK1 (F) and GAPDH protein expression levels. Graph shows GAPDH normalized AKT2 (B), P-AKT2 (C), NBA1 (D), SPK1 (E) and P-SPK1 (F) levels. Data are presented as the mean ± SEM; n=3. * P < 0.05, ** P < 0.01 compared with Con group; # P < 0.05, ## P < 0.01 compared with LPS induced group; NS=not significant.

Article Snippet: P-SPK1 (catalog number AP 05255PU-N) was from DBA Acris Antibodies (Rockville, MD, USA).

Techniques: Migration, Staining, Incubation, Western Blot, Expressing

Journal: Oncotarget

Article Title: Regulation of macrophage migration in ischemic mouse hearts via an AKT2/NBA1/SPK1 pathway

doi: 10.18632/oncotarget.23263

Figure Lengend Snippet: Mice were fed ATV (10 mg/kg/day) for 1 week before and after MI-injury. We produced MI animal model. Levels of P-AKT2 (A) , NBA1 z (B) , SPK1 (C) , P-SPK1 (D) , F4/80 (E) protein, F4/80 density (F) and ANP mRNA (G) increased following MI injury. ATV decreased protein levels of P-AKT2 (A), NBA1 (B), SPK1 (C), P-SPK1 (D), F4/80 (E), F4/80 density (F) and ANP mRNA (G) levels in WT MI animal model. Data are presented as the mean ± SEM; n=3. * P < 0.05, ** P < 0.01 compared with Con group; # P < 0.05, ## P < 0.01 compared with ATV treatment group; § P< 0.05, §§ P < 0.01 compared with MI group; NS=not significant.

Article Snippet: P-SPK1 (catalog number AP 05255PU-N) was from DBA Acris Antibodies (Rockville, MD, USA).

Techniques: Produced, Animal Model

Journal: Oncotarget

Article Title: Regulation of macrophage migration in ischemic mouse hearts via an AKT2/NBA1/SPK1 pathway

doi: 10.18632/oncotarget.23263

Figure Lengend Snippet: Mouse echocardiographic phenotype of WT vs SPK1−/−mice after MI 7 days

Article Snippet: P-SPK1 (catalog number AP 05255PU-N) was from DBA Acris Antibodies (Rockville, MD, USA).

Techniques:

Journal: Oncotarget

Article Title: Regulation of macrophage migration in ischemic mouse hearts via an AKT2/NBA1/SPK1 pathway

doi: 10.18632/oncotarget.23263

Figure Lengend Snippet: Organ weights and hemodynamics parameters of WT vs SPK1−/− mice after MI 7 days

Article Snippet: P-SPK1 (catalog number AP 05255PU-N) was from DBA Acris Antibodies (Rockville, MD, USA).

Techniques:

Journal: Oncotarget

Article Title: Regulation of macrophage migration in ischemic mouse hearts via an AKT2/NBA1/SPK1 pathway

doi: 10.18632/oncotarget.23263

Figure Lengend Snippet: WT and SPK1−/− mice were fed ATV (10 mg/kg/day) for 1 week before and after MI. Survival curve was observed between WT MI, WT + ATV MI, SPK1−/− MI, SPK1−/− + ATV MI groups. After MI 7 days, the death rate of SPK1−/− MI group is higher than that of WT MI group (A) . ATV treatment could decrease the death rate of WT MI model, but did not overtly lower the death rate of SPK1−/− models (A). * P<0.05, ** P<0.01 vs WT sham without ATV group; # P < 0.05, ## P < 0.01 compared with WT MI without ATV treatment group; § P < 0.05, §§ P < 0.01 compared with WT MI with ATV treatment group; ※ P>0.05 vs SPK1−/− MI without ATV treatment group. The infarction area of SPK1−/− MI group is bigger than that of WT MI group (B) . ATV could limit infarction area of WT MI with ATV treatment group (B). But there was no difference in the infarction area in SPK1−/− MI mice with and without ATV treatment (B). * P < 0.05, ** P < 0.01 compared with WT MI without ATV treatment group. # P< 0.05, ## P < 0.01 compared with WT MI with ATV treatment group. ATV could inhibit P-AKT2 (C) and NBA1 (D) protein levels in WT and SPK1−/− MI animal model. There is no difference in P-AKT2 and NBA1 protein level between WT MI and SPK1−/− MI with and without ATV treatment. ATV could inhibit F4/80 (E) protein levels, F4/80 density (F) , Ly6c density (G) , Ly6c mRNA (H) , flow cytometric analysis of Ly6c (I) , apoptosis density (J) , caspase 3 protein expression (K) , mRNA levels of ANP (L) , TNFα (M) , IL-6 (N) , collagen 1 (O) , collagen 3 (P) , collagen staining (Q) in the infarction area of WT MI mice. ATV could increase CD31 density (R) and CD 31 (S) mRNA level in WT MI model. But no difference in F4/80 (E) protein 6levels, F4/80 density (F), Ly6c density (G), Ly6c mRNA (H), flow cytometric analysis of Ly6c (I), apoptosis density (J), caspase 3 protein expression (K), mRNA levels of ANP (L), TNFα (M), IL-6 (N), collagen 1 (O), collagen 3 (P), collagen staining (Q), CD31 density (R), CD31(S) mRNA level were detected in SPK1−/− MI model with and without ATV treatment. Data are presented as the mean ± SEM. * P < 0.05, ** P < 0.01 compared with WT sham without ATV sham group; # P < 0.05, ## P < 0.01 compared with WT MI without ATV treatment group; § P < 0.05, §§ P < 0.01 compared with WT MI with ATV treatment group; † P < 0.05, †† P < 0.01 compared with SPK1−/− MI without ATV treatment group; NS=not significant.

Article Snippet: P-SPK1 (catalog number AP 05255PU-N) was from DBA Acris Antibodies (Rockville, MD, USA).

Techniques: Animal Model, Expressing, Staining

Journal: Oncotarget

Article Title: Regulation of macrophage migration in ischemic mouse hearts via an AKT2/NBA1/SPK1 pathway

doi: 10.18632/oncotarget.23263

Figure Lengend Snippet: LPS increased the protein expression levels of P-AKT2, NBA1, SPK1(P-SPK1) in LPS-induced macrophages. Myocardial infarction induced macrophages recruitment, apoptosis, inflammatory cytokine TNFα and IL-6 production in the infarction area. Moreover, ATV ameliorated myocradial remodeling by inhibiting macrophages recruitment, apoptosis, TNFα, IL-6, collagen I and III production and increasing CD31 expression in the infarction area.

Article Snippet: P-SPK1 (catalog number AP 05255PU-N) was from DBA Acris Antibodies (Rockville, MD, USA).

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 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: 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:

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: 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 reports . 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 reports . 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: A mammalian expression plasmid of N-terminal myc-FLAG-tagged human SPHK1 (GenBank data base accession number NM_021972) and one encoding Sp1 under the control of cytomegalovirus promoter were purchased from OriGene (Rockville, MD).

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: 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: A mammalian expression plasmid of N-terminal myc-FLAG-tagged human SPHK1 (GenBank data base accession number NM_021972) and one encoding Sp1 under the control of cytomegalovirus promoter were purchased from OriGene (Rockville, MD).

Techniques: 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: 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 (S1P 1 , S1P 2 , and S1P 3 ), and β-actin were quantitated by qRT-PCR (SYBR green assay), and the mRNA levels of SPHK1, SPHK2, S1P lyase, S1P 1 , S1P 2 , and S1P 3 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. Total cell lysates were collected 72 h after initiation of peretinoin treatment and transfection and used in an in vivo SPHK activity assay. Synthesized S1P was visualized by using radioisotopes. The radioactive spots corresponding to S1P were scraped into vials and the radioactivity was measured in a scintillation counter and normalized to that of DMSO-treated cells or empty vector-transfected cells. 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. ( d ), effects of peretinoin on the enzymatic activity of SPHK1 in vitro . Human recombinant SPHK1 was incubated with DMSO or peretinoin at 5, 10, 25, or 50 μM or with SKI II at 5, 10, 25, or 50 μM as described in the Experimental procedures. Synthesized S1P was quantitated by a fluorescence-based method. The SPHK activity in each condition was normalized to that of DMSO control. *p < 0.05, **p < 0.01, ***p < 0.005.

Article Snippet: A mammalian expression plasmid of N-terminal myc-FLAG-tagged human SPHK1 (GenBank data base accession number NM_021972) and one encoding Sp1 under the control of cytomegalovirus promoter were purchased from OriGene (Rockville, MD).

Techniques: Expressing, Activity Assay, Quantitative RT-PCR, SYBR Green Assay, Western Blot, Plasmid Preparation, Transfection, In Vivo, Synthesized, Radioactivity, Standard Deviation, In Vitro, Recombinant, Incubation, Fluorescence

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: 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 promoter activity of SPHK1 by Sp1 overexpression. A plasmid encoding Sp1 or a control empty vector was transfected into Huh-7 cells. After 24 h, the cells were transfected with a plasmid encoding luciferase under the control of the promoter region of SPHK1 or a control vector without any promoter regions. After another 24 h, DMSO or peretinoin at 20 or 40 μM was added. After 48-h DMSO or peretinoin treatment, luciferase activity was determined. This figure shows the relative ratio of the luciferase activity from peretinoin-treated cells to that of DMSO-treated cells in cells transfected with the plasmid encoding Sp1 or the control empty vector. 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. ( e ), attenuation of the suppressive effects of peretinoin on the promoter activity of SPHK1 by Sp1 knockdown. Three kinds of siRNA to Sp1, control siRNA (siCNT), or mock were transfected at 20 nM into Huh-7 cells and, 48 h later, the cells were transfected with the plasmid encoding luciferase under the control of the promoter region of SPHK1 or a control vector without any promoter regions. After another 24 h, DMSO or peretinoin at 40 μM was added. After 48-h DMSO or peretinoin treatment, luciferase activity was determined. This figure shows the relative ratio of the luciferase activity of cells treated with 40 μM peretinoin to that of DMSO-treated cells for each siRNA or mock transfection. ( f ), effects of deletion of the Sp1 binding site on the promoter activity of SPHK1. The plasmids encoding luciferase under the control of various lengths of promoter regions of SPHK1 were transfected into Huh-7 cells and, 24 h later, DMSO or peretinoin at 20 or 40 μM was added. After 48-h DMSO or peretinoin treatment, luciferase activity was determined. This figure shows the relative ratio of the luciferase activity of peretinoin-treated cells to that of DMSO-treated cells for each plasmid. Error bars indicate the standard deviation from three experiments and the statistical significance of the difference in the average was analyzed by two-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.005.

Article Snippet: A mammalian expression plasmid of N-terminal myc-FLAG-tagged human SPHK1 (GenBank data base accession number NM_021972) and one encoding Sp1 under the control of cytomegalovirus promoter were purchased from OriGene (Rockville, MD).

Techniques: Activity Assay, Luciferase, 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: 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: A mammalian expression plasmid of N-terminal myc-FLAG-tagged human SPHK1 (GenBank data base accession number NM_021972) and one encoding Sp1 under the control of cytomegalovirus promoter were purchased from OriGene (Rockville, MD).

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