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: 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

Journal: Biochemical and biophysical research communications

Article Title: Role of SK channel activation in determining the action potential configuration in freshly isolated human atrial myocytes from the SKArF study.

doi: 10.1016/j.bbrc.2019.03.074

Figure Lengend Snippet: Fig. 3. Presence of SK2 and SK3 channel subunits in human atrial myocytes. A. Confocal images (0.5-mm section) of a fixed and permeabilized cell labelled with anti-SK2 (green) and anti-SK3 (red). Labelling was observed at the cell periphery and superimposition of both images showed a significant degree of colocalization (yellow). B. Graph illustrates that an apparent similar level of labelling of SK2 and SK3 subunits in acutely isolated human atrial myocytes. C. Confocal images of SK2 (green) and SK3 (red) showing labelling at the cell edge. Superimposition of SK2 and SK3 channel subunit labelling demonstrates significant colocalization (yellow), which was quantified by using Mander's coefficient. Measured SK2 and SK3 labelling gave a Mander's coefficient of co-localization of SK3 overlapping SK2 by 0.94 ± 0.05 and SK2 overlapping SK3 by 093 ± 0.03. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Cells were then incubated overnight at room temperature with mouse anti-SK2 antibody (Biorbyt: orb333955) and rabbit anti-SK3 antibody (Biorbyt: orb157735), added as a 1:200 dilution in blocking solution of composition (mM): phosphate buffer saline, bovine serum albumin (Sigma Aldrich, A9418) (1%), and foetal calf serum (Thermo Fisher Scientific, 10500-064) (2%) [20,21].

Techniques: Isolation

Journal: Biomolecules & biomedicine

Article Title: Deletion of sphingosine kinase 2 attenuates cigarette smoke-mediated chronic obstructive pulmonary disease-like symptoms by reducing lung inflammation.

doi: 10.17305/bjbms.2022.8034

Figure Lengend Snippet: Figure 2. CS-induced pulmonary fibrosis was alleviated in SphK2−/−

Article Snippet: The primary antibodies specific for SphK2 (A01382-1, Boster Biol.

Techniques:

Journal: Biomolecules & biomedicine

Article Title: Deletion of sphingosine kinase 2 attenuates cigarette smoke-mediated chronic obstructive pulmonary disease-like symptoms by reducing lung inflammation.

doi: 10.17305/bjbms.2022.8034

Figure Lengend Snippet: Figure 1. Increased SphK2 caused by chronic CS exposure in the lung tissue of WT mice. (A) Mice were subjected to CS exposure 5 days a week for 6 months; the SphK2 mRNA levels in the lung tissues were measured and (B) quantified by RT-PCR at indicated times (0, 0.5, 1, 2, 4 and 6 months) after CS exposure; (C) The SphK2 protein levels in the lung tissue were measured and (D) quantified by western blot at indicated times (0, 1, 2, 3, 4 and 6 months) after CS exposure (n = 4, §P < 0.05, vs 0-month group); (E) The S1P levels in the collected BALF were measured by ELISA (n = 4, ∗P < 0.05, vs non-CS exposure; P < 0.05, vs CS exposure for 30 days); (F) Quantification of SphK2 mRNA levels in lung, heart, liver, and kidney of mice exposed to air or CS for six months (n = 5, **P < 0.05, vs control group (Con) of lung, heart, liver, or kidney tissue). SphK2: Sphingosine kinase 2; CS: Cigarette smoke; WT: Wild-type; RT-PCR: Real-time polymerase chain reaction; BALF: Bronchoalveolar lavage fluid; GADPH: Glyceraldehyde-3- phosphate dehydrogenase.

Article Snippet: The primary antibodies specific for SphK2 (A01382-1, Boster Biol.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Control, Real-time Polymerase Chain Reaction

Journal: Biomolecules & biomedicine

Article Title: Deletion of sphingosine kinase 2 attenuates cigarette smoke-mediated chronic obstructive pulmonary disease-like symptoms by reducing lung inflammation.

doi: 10.17305/bjbms.2022.8034

Figure Lengend Snippet: Figure 3. CS-induced pulmonary inflammation was attenuated in SphK2−/−mice. WT and SphK2−/−mice were both subjected to air or CS exposure 5 days a week for 6 months. The BALF was collected from mice of both genotypes and analyzed by flow cytometry. (A) BALF cells were sorted by FACS with anti-GFP antibody, followed by gating with anti-CD45 antibody; (B) Representative cytoflow sorting images show the proportion of CD45+CD11b+

Article Snippet: The primary antibodies specific for SphK2 (A01382-1, Boster Biol.

Techniques: Cytometry

Journal: Biomolecules & biomedicine

Article Title: Deletion of sphingosine kinase 2 attenuates cigarette smoke-mediated chronic obstructive pulmonary disease-like symptoms by reducing lung inflammation.

doi: 10.17305/bjbms.2022.8034

Figure Lengend Snippet: Figure 4. Reduced CFTR expression and attenuation of pulmonary inflammation and fibrosis in CS-exposed SphK2−/−mice. (A) Western blotting analysis showed that the protein expression of CFTR, phospho-Nf-κB-p65, and GAPDH in lung tissues of WT mice and SphK2−/−mice under normal air or CS exposure; (B-C) Quantification of the relative protein expression of CFTR and phospho-Nf-κB-p65 (n = 4, ∗P < 0.05, vs NA-exposed WT mice; **P < 0.05, vs CS-exposed WT mice); (D) Immunofluorescence staining of elastin (green)/phospho-Nf-κB-p65 (red)/Dapi (blue) of lung tissue sections from CS-exposed WT mice and SphK2−/−mice; (E) Representative cytoflow sorting images show the proportion of CD45+CD11b+ cells in BALF of SphK2−/−mice treated with or without S1P1 agonist; (F-G) Quantification of the relative protein expression of CFTR and phospho-Nf-κB-p65 in lung tissues of SphK2−/−

Article Snippet: The primary antibodies specific for SphK2 (A01382-1, Boster Biol.

Techniques: Expressing, Western Blot, Staining

Journal: Biomolecules & biomedicine

Article Title: Deletion of sphingosine kinase 2 attenuates cigarette smoke-mediated chronic obstructive pulmonary disease-like symptoms by reducing lung inflammation.

doi: 10.17305/bjbms.2022.8034

Figure Lengend Snippet: Figure 5. FTY720 treatment rescued CFTR function and attenuated pulmonary inflammation and fibrosis in CS-exposed mice. WT mice treated with or without FTY720 were both exposed to CS 5 days a week for 6 months. (A) Western blotting analysis reveals the protein levels of CFTR and phospho- Nf-κB-p65 in lung tissues of mice; (B-C) Quantification of the relative protein expression of CFTR and phospho-Nf-κB-p65 in lung tissues of mice (n = 4, ∗P < 0.05, vs control mice; #P < 0.05, vs CS-exposed WT mice); (D) Masson’s trichrome staining shows the alveolar structure (red) and collagen deposition (blue) in lung tissues of mice; (E) The alveolar chord length was measured on HE-stained lung sections (n = 10, ∗P < 0.05, vs control mice; #P < 0.05, vs CS-exposed WT mice); (F) Quantification (%) of small airway collagen deposition (n = 4, ∗P < 0.05, vs control mice; #P < 0.05, vs CS-exposed WT mice); (G) Representative cytoflow sorting images show the proportion of CD45+CD11b+ subset in BALF of mice; (H-I) Quantification of the proportions and the number of CD45+CD11b+ subsets (n = 5, ∗P < 0.05, vs control mice; #P < 0.05, vs CS-exposed WT mice). SphK2: Sphingosine kinase 2; CS: Cigarette smoke; WT: Wild-type; BALF: Bronchoalveolar lavage fluid; GADPH: Glyceraldehyde-3-phosphate dehydrogenase; CFTR: Cystic fibrosis transmembrane conductance regulator.

Article Snippet: The primary antibodies specific for SphK2 (A01382-1, Boster Biol.

Techniques: Western Blot, Expressing, Control, Staining

Journal: Biomolecules & biomedicine

Article Title: Deletion of sphingosine kinase 2 attenuates cigarette smoke-mediated chronic obstructive pulmonary disease-like symptoms by reducing lung inflammation.

doi: 10.17305/bjbms.2022.8034

Figure Lengend Snippet: Figure 6. Schematic diagram showing signaling pathways involved in SphK2-induced pulmonary inflammation and fibrosis after chronic CS exposure. SphK2: Sphingosine kinase 2; CS: Cigarette smoke; WT: Wild-type; CFTR: cystic fibrosis transmembrane conductance regulator; IL: Interleukin.

Article Snippet: The primary antibodies specific for SphK2 (A01382-1, Boster Biol.

Techniques: Protein-Protein interactions

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