Journal: PLoS ONE

Article Title: The Molecular Mechanism of Amyloid β42 Peptide Toxicity: The Role of Sphingosine Kinase-1 and Mitochondrial Sirtuins

doi: 10.1371/journal.pone.0137193

Figure Lengend Snippet: PC12 cells were incubated in the presence of oligomeric Aβ (AβO, 1 μM) for 24 and 96 h. (A–B) The activity of Sphk1 and Sphk2 was determined, as described in Methods. (C–F) The levels of mRNA of Sphk1 and Sphk2 genes were analysed by quantitative RT-PCR. The results of RT-PCR were normalized to Actb gene expression and are presented as the mean ± SEM from 4 independent experiments; *, **, *** for p < 0.05, 0.01, and 0.001, respectively, as compared with the corresponding control, by using Student’s t-test.

Article Snippet: The level of mRNA for selected genes was analysed by using TaqMan Gene Expression Assays according to the manufacturer’s instructions: ACTB-4352340E ( Actb ), Rn00591307-m1 ( Sphk1 ), Rn01457923-g1 ( Sphk2 ), Rn99999125_m1 ( Bcl2 l), Rn01501410-m1 ( Sirt3 ), Rn01481485-m1 ( Sirt4 ), Rn01450559-m1 ( Sirt5 ), Rn01475306-m1 ( Aifm1 ), Rn00565018-m1 ( Parp1 ), Rn00560930_m1 ( Cat ), Rn00755717_m1 ( Tp53 ).

Techniques: Incubation, Activity Assay, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Control

Journal: Scientific reports

Article Title: A multi-omics approach identifies the key role of disorders of sphingolipid metabolism in Ang II-induced hypertensive cardiomyopathy myocardial remodeling.

doi: 10.1038/s41598-024-81611-8

Figure Lengend Snippet: Fig. 10. Experimental validation. (A) Contents of S1P in myocardium. (B) Contents of S1P in plasma. (C) Representative Western blot. (D–I) Expression of SPHK1, SPHK2, S1PR1, HIF-1α, TGF-β, and FOXO1 relative to GAPDH levels. One target protein corresponds to one gel in the figure, and complete gel and blot images are provided in detail in the Supplementary file. Data are mean ± SD, n = 3–6. **P < 0.01, ***P < 0.001.

Article Snippet: Subsequently, the membrane was incubated in 5% skimmed milk at 25 °C for 1 h. Then, the membranes were washed and incubated overnight at 4 °C with the following primary antibodies: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cat No. ET1601-4, HUABIO, China), Sphingosine kinase 1 (SPHK1) (Cat No. 10670- 1-AP, Proteintech, Wuhan, China), Sphingosine kinase 2 (SPHK2) (Cat No. 21898-1-AP, Proteintech, Wuhan, China), Hypoxia Inducible Factor-1α (HIF-1α) (Cat No. ab179483, Abcam, Cambridge, United Kingdom), Transforming growth factor-β (TGF-β) (Cat No. 21898-1-AP, Proteintech, Wuhan, China), Forkhead Box O1 (FOXO1) (Cat No. ET1608-25, HUABIO, China).

Techniques: Biomarker Discovery, Clinical Proteomics, Western Blot, Expressing

Journal: Human Molecular Genetics

Article Title: The mutant Moonwalker TRPC3 channel links calcium signaling to lipid metabolism in the developing cerebellum

doi: 10.1093/hmg/ddv150

Figure Lengend Snippet: Significant expression changes (≥1.5-fold) in Mwk Purkinje cells compared with wild-type littermates in genes associated with lipid metabolism ( P ≤ 0.05)

Article Snippet: The following antibodies were used: p44/42 MAPK (Erk1/2) (Cell Signaling; 1:1000), phospho-p44/42 MAPK (Erk1/2) (Tyr202/Tyr204) (Cell Signaling; 1:1000), CaMKIV (Abcam; 1:1000), p-CaMKIV (Thr196) (Santa Cruz; 1:1000), CaMKII (Cell Signaling, 1:500), phospho-CaMKII (Cell Signaling, 1:1000), CREB (Cell Signaling, 1:500), phospho-CREB (Ser133) (Cell Signaling, 1:1000), ASAH1 (Santa Cruz, 1:200), SPHK2 (Novus, 1:500), SGPP1 (Novus, 1:1000), SMPD1 (Santa Cruz, 1:200), SGMS1 (Santa Cruz, 1:200), Actin (Abcam, 1:1000).

Techniques: Expressing, Binding Assay, Derivative Assay

Journal: Human Molecular Genetics

Article Title: The mutant Moonwalker TRPC3 channel links calcium signaling to lipid metabolism in the developing cerebellum

doi: 10.1093/hmg/ddv150

Figure Lengend Snippet: Protein expression changes in enzymes involved in ceramide homeostasis. ( A ) Schematic diagram of mammalian ceramide biosynthesis. Differentially expressed enzymes in Mwk Purkinje cells are indicated in red (upregulated) and blue (downregulated). ( B ) Cerebellar extracts from 3-week-old mutant Mwk mice and WT littermates were subjected to immunoblotting for lipid metabolism enzymes. ASAH1 and SPHK2 levels were increased in the Mwk cerebellum (left panel), whereas levels of SGPP1, SMPD1 and SGMS1 were reduced in Mwk cerebellum (middle panel). Actin levels are included as loading control. Densitometry quantifications are shown (right panel). Statistical significance was determined by one-way ANOVA ( n = 3). ** P < 0.01; *** P < 0.001.

Article Snippet: The following antibodies were used: p44/42 MAPK (Erk1/2) (Cell Signaling; 1:1000), phospho-p44/42 MAPK (Erk1/2) (Tyr202/Tyr204) (Cell Signaling; 1:1000), CaMKIV (Abcam; 1:1000), p-CaMKIV (Thr196) (Santa Cruz; 1:1000), CaMKII (Cell Signaling, 1:500), phospho-CaMKII (Cell Signaling, 1:1000), CREB (Cell Signaling, 1:500), phospho-CREB (Ser133) (Cell Signaling, 1:1000), ASAH1 (Santa Cruz, 1:200), SPHK2 (Novus, 1:500), SGPP1 (Novus, 1:1000), SMPD1 (Santa Cruz, 1:200), SGMS1 (Santa Cruz, 1:200), Actin (Abcam, 1:1000).

Techniques: Expressing, Mutagenesis, Western Blot

Journal: Oncotarget

Article Title: TNFα mediated ceramide generation triggers cisplatin induced apoptosis in B16F10 melanoma in a PKCδ independent manner

doi: 10.18632/oncotarget.26478

Figure Lengend Snippet: (A) B16F10 cells were transfected with PKCδ siRNA followed by cisplatin treatment in a time dependent manner. At different time points the treated cells were collected in TRIZOL for Sphk1 and Sphk2 mRNA expression by semi quantitative RT-PCR analysis. GAPDH was used as a reference. Data are from one of the three representative experiments. At different time points, data were represented as mean ± SD. * P < 0.05, ** P < 0.001 vs untreated. (B) B16F10 cells were transfected with PKCδ siRNA followed by cisplatin treatment. Activities of sphingosine kinase 1 and sphingosine kinase 2 were determined from cell lysates following the protocol described in Materials and Methods. Bar diagram is represented as mean ± SD. * P < 0.05, ** P < 0.001 vs untreated. (C) In a separate experiment, similarly PKCδ silenced cisplatin treated B16F10 cells were transfected with Sphk1 and Sphk2 specific siRNAs as mentioned in Materials and Methods. Cells were collected stained with Annexin-V FITC and PI and subsequently analyzed by flow cytometry. Data are from one of the three representative experiments.

Article Snippet: Mouse specific IRF1 small-interfering RNA (siRNA), human specific PKCδ, IRF1, Sphk2 siRNA and control siRNAs were obtained from Santa Cruz Biotechnology.

Techniques: Transfection, Expressing, Quantitative RT-PCR, Staining, Flow Cytometry

Journal: Oncotarget

Article Title: TNFα mediated ceramide generation triggers cisplatin induced apoptosis in B16F10 melanoma in a PKCδ independent manner

doi: 10.18632/oncotarget.26478

Figure Lengend Snippet: (A) 2 × 10 6 A375 cells were transfected with PKCδ specific siRNA or control siRNA as mentioned in Materials and Methods and the transfected cells were collected in TRIZOL for mRNA expression of PKCδ by semi quantitative RT-PCR. GAPDH was used as a reference. Data are from one of the three representative experiments. Bar diagram is represented as mean ± SD. * P < 0.05, ** P < 0.001 vs untreated. (B) In a separate experiment, similarly A375 cells were either transfected with PKCδ specific siRNA or control siRNA as mentioned in Materials and Methods. The expression of PKCδ was analyzed in whole cell lysates by western blot analysis. GAPDH was used as a reference. Data are from one of the three representative experiments. Bar diagram is represented as mean ± SD. * P < 0.05, ** P < 0.001 vs untreated. (C) PKCδ silenced A375 cells were either treated with cisplatin alone or in combination with imipramine, ATK or transfected with IRF1 specific siRNAs. Cells were also treated with exogenous TNFα in absence of cisplatin. Treated cells were subsequently stained with anti-mouse ceramide-FITC, isotype-matched control mouse antibody. Ceramide expression was analysed by flow cytometry. Data are from one of the three representative experiments. Bar diagram is represented as mean ± SD. * P < 0.05, ** P < 0.001 vs untreated. (D) PKCδ silenced A375 cells were either treated with cisplatin alone or in combination with imipramine, ATK, TNFα/TNFα-R1 neutralizing antibody or transfected with IRF1/Sphk2 specific siRNAs as mentioned in Materials and Methods. Cells were collected, stained with Annexin-V FITC and PI and subsequently analyzed by flow cytometry. Bar diagram is represented as mean ± SD. * P < 0.05, ** P < 0.001 vs untreated. (E) and (F) A375 cells, transfected with PKCδ siRNA followed by cisplatin treatment were cultured in hypoxic chamber as described in Materials and Methods. Cells were collected and subsequently stained with anti-mouse ceramide-FITC, isotype-matched control mouse antibody. Ceramide expression was analysed by flow cytometry. In a different experiment cells were stained with Annexin-V FITC and PI and subsequently analyzed by flow cytometry. Data are from one of the three representative experiments.

Article Snippet: Mouse specific IRF1 small-interfering RNA (siRNA), human specific PKCδ, IRF1, Sphk2 siRNA and control siRNAs were obtained from Santa Cruz Biotechnology.

Techniques: Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot, Staining, Flow Cytometry, Cell Culture

Journal: Oncotarget

Article Title: TNFα mediated ceramide generation triggers cisplatin induced apoptosis in B16F10 melanoma in a PKCδ independent manner

doi: 10.18632/oncotarget.26478

Figure Lengend Snippet: The primer sequences

Article Snippet: Mouse specific IRF1 small-interfering RNA (siRNA), human specific PKCδ, IRF1, Sphk2 siRNA and control siRNAs were obtained from Santa Cruz Biotechnology.

Techniques: Sequencing

Journal: ACS Infectious Diseases

Article Title: Sphingosine Kinases Promote Ebola Virus Infection and Can Be Targeted to Inhibit Filoviruses, Coronaviruses, and Arenaviruses Using Late Endocytic Trafficking to Enter Cells

doi: 10.1021/acsinfecdis.2c00416

Figure Lengend Snippet: Study of the relationship between antiviral and SK1/2 inhibitory activities using derivatives of PF-543. SK1 and SK2 activity was assessed by measuring fluorescence after incubation of PF-543 derivatives (1 μM) or vehicle (DMSO, 0.1%) with SK reaction buffers containing baculovirus-derived recombinant SK1 or SK2 and light-sensitive NBD-sphingosine. Percent SK activity was calculated in comparison to the vehicle, which represents 100% activity. In parallel, antiviral activity was determined by treating HT1080 cells with the PF-543 derivatives (10 μM) or vehicle (0.1%) and transducing them with MLV pseudotypes encoding LacZ and harboring EBOV GPΔmuc. Relative antiviral activity (%) was determined by quantifying LacZ-positive inhibitor-treated cells compared to vehicle-treated cells. Mean SK1 activity ( x axis) and SK2 activity ( y axis) was plotted as a bubble chart using R studio with the size of the bubble representing mean antiviral activity.

Article Snippet: Primary antibodies used were NPC1 (ab134113, Abcam), Akt (9272S, Cell Signaling Technology), phospho-Akt S473 (92721S, Cell Signaling Technology), GAPDH (ab8245, Abcam), SK1 (12071S, Cell Signaling Technology), SK2 (32346S, Cell Signaling Technology), and pan-filovirus anti-GP antibody (21D10, IBT Bioservices).

Techniques: Activity Assay, Fluorescence, Incubation, Derivative Assay, Recombinant, Comparison

Journal: ACS Infectious Diseases

Article Title: Sphingosine Kinases Promote Ebola Virus Infection and Can Be Targeted to Inhibit Filoviruses, Coronaviruses, and Arenaviruses Using Late Endocytic Trafficking to Enter Cells

doi: 10.1021/acsinfecdis.2c00416

Figure Lengend Snippet: SK1/2 knockdown blocks EBOV GP-mediated entry in HT1080 cells. HT1080 cells transfected with nontargeting control DsiRNA (NC: negative control) or DsiRNA targeting SK1 or SK2 were either (A) lysed and expression of SK1 or SK2 (upper) and Vinculin (lower) detected by immunoblotting or (B) infected with βlam VLPs harboring EBOV GPΔmuc or VSV G. Relative entry % was determined by measuring the percentage of inhibitor-treated cells with cleaved βlam substrate (CCF2) compared to vehicle-treated cells. Results are expressed as mean ± s.d. of triplicates and are representative of three experiments. Variance was analyzed by one-way ANOVA, followed by Dunnett’s multiple comparison test to determine significance. *** p < 0.001.

Article Snippet: Primary antibodies used were NPC1 (ab134113, Abcam), Akt (9272S, Cell Signaling Technology), phospho-Akt S473 (92721S, Cell Signaling Technology), GAPDH (ab8245, Abcam), SK1 (12071S, Cell Signaling Technology), SK2 (32346S, Cell Signaling Technology), and pan-filovirus anti-GP antibody (21D10, IBT Bioservices).

Techniques: Knockdown, Transfection, Control, Negative Control, Expressing, Western Blot, Infection, Comparison

Journal: Cellular signalling

Article Title: Metastatic triple-negative breast cancer is dependent on SphKs/S1P signaling for growth and survival

doi: 10.1016/j.cellsig.2017.01.021

Figure Lengend Snippet: Human breast cancer MCF-7 cells (A, B), human TNBC MDA-MB-231 (C), and human lung metastatic LM2-4 cells originated from MDA-MB-231 cells (B) were cultured in 1% serum. Cells were pre-treated with 5 μM (A) and 10 μM (B, C, D) PF543 (SphK1 inhibitor), K145 (SphK2 inhibitor), and DMS (SphKs inhibitor) before treatment of 100 ng/ml EGF for 3 days. Cells growth were measured with WST-8 regents according to the manufacture’s protocol. Optical density was measured at 450 nm using Biotek plate reader. Back ground corrected optical densities were plotted. Similar results were obtained in two additional experiments.

Article Snippet: SphK2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001204160","term_id":"345197211","term_text":"NM_001204160"}} NM_001204160 ) human cDNA expression vector (pCMV6-SphK2) was from OriGene Technologies (Rockville, MD, USA).

Techniques: Cell Culture

Journal: Cellular signalling

Article Title: Metastatic triple-negative breast cancer is dependent on SphKs/S1P signaling for growth and survival

doi: 10.1016/j.cellsig.2017.01.021

Figure Lengend Snippet: Human breast cancer MCF-7 cells (A), human TNBC MDA-231 (C), and human lung metastatic LM-2-4 cells originated from MDA-231 cells (B) were transfected with control siRNA and siRNAs targeted to SphKs for 48 hours. Serum starved cells were treated with or without 100 ng/ml EGF for 15 minutes. Levels of p-ERK, p-AKT, p-p38, p-p70S6K, SphK1 proteins in the nuclei-free extracts were analyzed by immunoblotting with the indicated antibodies. Blots were stripped and re-probed with anti-α-tubulin antibody to show equal loading and transfer. Level of SphK2 protein in the nuclear extract was analyzed by immunoblotting with anti-SphK2 antibodies. Nuclear extract from MCF-7 cells transiently transfected with untagged SphK2 was included to indicate the molecular weight of this protein [20]. Blots were stripped and re-probed with anti-lamin A/C antibody to show equal loading and transfer. Similar results were obtained in two additional experiments.

Article Snippet: SphK2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001204160","term_id":"345197211","term_text":"NM_001204160"}} NM_001204160 ) human cDNA expression vector (pCMV6-SphK2) was from OriGene Technologies (Rockville, MD, USA).

Techniques: Transfection, Western Blot, Molecular Weight

Journal: Cellular signalling

Article Title: Metastatic triple-negative breast cancer is dependent on SphKs/S1P signaling for growth and survival

doi: 10.1016/j.cellsig.2017.01.021

Figure Lengend Snippet: Human breast cancer MCF-7 cells (A), human TNBC MDA-MB-231 (C), and human lung metastatic LM-2-4 cells originated from MDA-MB-231 cells (B) were transfected with control siRNA and siRNAs targeted to SphKs for 24 hours. Cells were grown in 1% serum containing medium and treated with or without EGF (100 ng/ml) for 3 days. Cells growth was measured with WST-8 regents according to the manufacture’s protocol. Optical density was measured at 450 nm using Biotek plate reader. Back ground corrected optical densities were plotted. Similar results were obtained in two additional experiments. Downregulation of SphK1 and SphK2 with siRNAs (D). RNA was isolated and mRNA levels of SphK1 and SphK2, and Cyclophilin A mRNAs were determined by quantitative real-time PCR.

Article Snippet: SphK2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001204160","term_id":"345197211","term_text":"NM_001204160"}} NM_001204160 ) human cDNA expression vector (pCMV6-SphK2) was from OriGene Technologies (Rockville, MD, USA).

Techniques: Transfection, Isolation, Real-time Polymerase Chain Reaction

Journal: Cellular signalling

Article Title: Metastatic triple-negative breast cancer is dependent on SphKs/S1P signaling for growth and survival

doi: 10.1016/j.cellsig.2017.01.021

Figure Lengend Snippet: LM2-4, MDA-MB-231, and MCF-7 cells were grown in 2% serum containing medium for 24 hours. Cells were scratched with a pipette tip and pre-treated with 2 μM PF543 (SphK1 inhibitor), and K145 (SphK2 inhibitor before treatment of EGF (100ng/ml) for 8 hours (A.B) and 24 hours (C). Migrating cells were photographed under a phase contrast microscope.

Article Snippet: SphK2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001204160","term_id":"345197211","term_text":"NM_001204160"}} NM_001204160 ) human cDNA expression vector (pCMV6-SphK2) was from OriGene Technologies (Rockville, MD, USA).

Techniques: Transferring, Microscopy

Journal: Cellular signalling

Article Title: Metastatic triple-negative breast cancer is dependent on SphKs/S1P signaling for growth and survival

doi: 10.1016/j.cellsig.2017.01.021

Figure Lengend Snippet: LM2-4, MDA-MB-231, and MCF-7 cells were transfected with control siRNA, siRNA targeted to SphK1 or SphK2 for 24 hours. Cells were grown in 1% serum containing medium for another 24 hours. Cells were scratched with a pipette tip and then incubated with EGF (100 ng/ml) for 24 hours (A, B) and 48 hours (C). Migrating cells were photographed under a phase contrast microscope.

Article Snippet: SphK2 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_001204160","term_id":"345197211","term_text":"NM_001204160"}} NM_001204160 ) human cDNA expression vector (pCMV6-SphK2) was from OriGene Technologies (Rockville, MD, USA).

Techniques: Transfection, Transferring, Incubation, Microscopy