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: Applied and Environmental Microbiology

Article Title: Isolation and Characterization of a Mutant of the Marine Bacterium Alcanivorax borkumensis SK2 Defective in Lipid Biosynthesis

doi: 10.1128/aem.02832-09

Figure Lengend Snippet: FIG. 1. TLC analysis of lipids accumulated in cells of A. borkumen- sis SK2 and mutant strains. Cells were cultivated aerobically in ONR7a medium containing 1% (wt/vol) sodium pyruvate at 30°C and 150 rpm. After 72 h of cultivation, cells were harvested, washed, and lyophilized and the lipids were extracted with chloroform-methanol (2:1, vol/vol). Lipid extracts obtained from 5 mg lyophilized cells were applied in each lane. The solvent system used for elution was hexane-diethyl ether-acetic acid (90:7.5:1, vol/vol/vol), and lipids were visualized by stain- ing with iodine vapors. Lanes: T, triolein; OA, oleic acid; OO, oleyl oleate; WT, A. borkumensis SK2; atfA1 atfA2, A. borkumensis atfA1Km atfA2Sm; K, control (“culture” without cells); and 2 M21 to 2 M131, different mini-Tn5 transposon-induced mutants. FA, fatty acids.

Article Snippet: Bacterial strains and plasmids used in this study Strain or plasmid Relevant characteristicsa Source and/orreference A. borkumensis strains SK2 (DSM 11573) Type strain; wild type; TAG and WE producer 49; DSMZ atfA1 Km mutant atfA1 disruption mutant; Kmr 28 atfA2 Sm mutant atfA2 disruption mutant; Smr 28 atfA1 Km atfA2 Sm double mutant atfA1 atfA2 double disruption mutant; Kmr Smr 28 ABO_1185 Sm mutant ABO_1185 disruption mutant; Smr; derivative of SK2 This study E. coli strains SM10 ( pir) Apr Cmr Kmr pUTmini-Tn5Cm 13 TOP10 F mcrA (mrr-hsdRMS-mcrBC) rpcL nupG endA1 deoR 80lacZ M15 lacX74 recA1 araD139 (ara-leu)7697 galU galK Invitrogen S17-1 recA1 thi-1 hsdR17(rK mK ) proA tra-RP4 40 Plasmids pBluescript SK( ) Plasmid used for subcloning of DNA; Apr lacPOZ Stratagene pUTmini-Tn5Cm Suicide vector for mutagenesis with mini-Tn5; Cmr 13 pCR2.1-TOPO Plasmid used for subcloning of DNA; Apr Invitrogen pCR2.1-TOPO::ABO_1185 Derivative of pCR2.1-TOPO containing 1,908-bp ABO_1185 gene This study pCR2.1-TOPO::ABO_1185 Sm Sm cassette cloned into NcoI site of pCR2.1-TOPO::ABO_1185; Apr Smr This study pSUP202 Apr Cmr Tcr; carries ColE1 origin and mob site; unable to replicate in A. borkumensis 40 pSUP202::ABO_1185 Sm Fusion of pSUP202 and ABO_1185 Sm fragment of pCR2.1-TOPO via BamHI/HindIII sites; carries mob site; Cmr Apr Smr This study a For abbreviations for genotypes of E. coli, see reference 7.

Techniques: Mutagenesis, Solvent, Staining, Control

Journal: Applied and Environmental Microbiology

Article Title: Isolation and Characterization of a Mutant of the Marine Bacterium Alcanivorax borkumensis SK2 Defective in Lipid Biosynthesis

doi: 10.1128/aem.02832-09

Figure Lengend Snippet: FIG. 2. Characterization of mutant 2 M131 with regard to lipid formation. (A) TLC analysis of storage lipid accumulation in cells of A. borkumensis SK2 and of mutant 2 M131. Cells were cultivated for 72 h in ONR7a medium containing 1% (wt/vol) sodium pyruvate, and lipid extracts were analyzed by TLC. Lipid extracts were obtained from 3 mg freeze-dried cells and the corresponding amount of the supernatant. Triolein (T), oleic acid (OA), and oleyl oleate (OO) were used as standards. Lanes: SK2 C, extract from cells of A. borkumensis SK2; 2 M131 C, extract from cells of the mutant 2 M131; SK2 S, extract from a supernatant sample from the A. borkumensis SK2 culture; and 2 M131 S, extract from a supernatant sample from the mutant 2 M131 culture. FA, fatty acids. (B) Organization of the mini-Tn5 transposon insertion site and the adjacent region in the genome of mutant 2 M131. The insertion site was mapped to a gene coding for a cytochrome c family protein (ABO_1185) (39). ABO_1182, gene for hypothetical protein; ABO_1183, gene for putative aminotransferase; ABO_1184, gene for hypothetical protein; ABO_1185, gene for cytochrome c family protein; ABO_1186, gene for CorA-like Mg2 transporter protein; ABO_1187, gene for hypothetical protein; cysI, sulfite reductase (NADPH) gene. The insertion site is shown by an inverted triangle.

Article Snippet: Bacterial strains and plasmids used in this study Strain or plasmid Relevant characteristicsa Source and/orreference A. borkumensis strains SK2 (DSM 11573) Type strain; wild type; TAG and WE producer 49; DSMZ atfA1 Km mutant atfA1 disruption mutant; Kmr 28 atfA2 Sm mutant atfA2 disruption mutant; Smr 28 atfA1 Km atfA2 Sm double mutant atfA1 atfA2 double disruption mutant; Kmr Smr 28 ABO_1185 Sm mutant ABO_1185 disruption mutant; Smr; derivative of SK2 This study E. coli strains SM10 ( pir) Apr Cmr Kmr pUTmini-Tn5Cm 13 TOP10 F mcrA (mrr-hsdRMS-mcrBC) rpcL nupG endA1 deoR 80lacZ M15 lacX74 recA1 araD139 (ara-leu)7697 galU galK Invitrogen S17-1 recA1 thi-1 hsdR17(rK mK ) proA tra-RP4 40 Plasmids pBluescript SK( ) Plasmid used for subcloning of DNA; Apr lacPOZ Stratagene pUTmini-Tn5Cm Suicide vector for mutagenesis with mini-Tn5; Cmr 13 pCR2.1-TOPO Plasmid used for subcloning of DNA; Apr Invitrogen pCR2.1-TOPO::ABO_1185 Derivative of pCR2.1-TOPO containing 1,908-bp ABO_1185 gene This study pCR2.1-TOPO::ABO_1185 Sm Sm cassette cloned into NcoI site of pCR2.1-TOPO::ABO_1185; Apr Smr This study pSUP202 Apr Cmr Tcr; carries ColE1 origin and mob site; unable to replicate in A. borkumensis 40 pSUP202::ABO_1185 Sm Fusion of pSUP202 and ABO_1185 Sm fragment of pCR2.1-TOPO via BamHI/HindIII sites; carries mob site; Cmr Apr Smr This study a For abbreviations for genotypes of E. coli, see reference 7.

Techniques: Mutagenesis

Journal: Applied and Environmental Microbiology

Article Title: Isolation and Characterization of a Mutant of the Marine Bacterium Alcanivorax borkumensis SK2 Defective in Lipid Biosynthesis

doi: 10.1128/aem.02832-09

Figure Lengend Snippet: FIG. 3. TLC analysis of lipid biosynthesis by A. borkumensis SK2, mutant 2 M131, and the constructed A. borkumensis ABO_1185Sm mutant. Cells were cultivated aerobically in ONR7a medium contain- ing 1% (wt/vol) sodium pyruvate at 30°C and 150 rpm. After 72 h of cultivation, cells were harvested, washed, and lyophilized and the lipids were extracted with chloroform-methanol (2:1, vol/vol). Lipid extracts obtained from 5 mg lyophilized cells were applied in each lane. The solvent system used for elution was hexane-diethyl ether-acetic acid (80:20:1, vol/vol/vol), and lipids were visualized by staining with iodine vapor. Lanes: T, triolein; OA, oleic acid; OO, oleyl oleate; SK2, A. borkumensis wild type; 2 M131, transposon-induced mutant; ABO_1185, A. borkumensis ABO_1185Sm.

Article Snippet: Bacterial strains and plasmids used in this study Strain or plasmid Relevant characteristicsa Source and/orreference A. borkumensis strains SK2 (DSM 11573) Type strain; wild type; TAG and WE producer 49; DSMZ atfA1 Km mutant atfA1 disruption mutant; Kmr 28 atfA2 Sm mutant atfA2 disruption mutant; Smr 28 atfA1 Km atfA2 Sm double mutant atfA1 atfA2 double disruption mutant; Kmr Smr 28 ABO_1185 Sm mutant ABO_1185 disruption mutant; Smr; derivative of SK2 This study E. coli strains SM10 ( pir) Apr Cmr Kmr pUTmini-Tn5Cm 13 TOP10 F mcrA (mrr-hsdRMS-mcrBC) rpcL nupG endA1 deoR 80lacZ M15 lacX74 recA1 araD139 (ara-leu)7697 galU galK Invitrogen S17-1 recA1 thi-1 hsdR17(rK mK ) proA tra-RP4 40 Plasmids pBluescript SK( ) Plasmid used for subcloning of DNA; Apr lacPOZ Stratagene pUTmini-Tn5Cm Suicide vector for mutagenesis with mini-Tn5; Cmr 13 pCR2.1-TOPO Plasmid used for subcloning of DNA; Apr Invitrogen pCR2.1-TOPO::ABO_1185 Derivative of pCR2.1-TOPO containing 1,908-bp ABO_1185 gene This study pCR2.1-TOPO::ABO_1185 Sm Sm cassette cloned into NcoI site of pCR2.1-TOPO::ABO_1185; Apr Smr This study pSUP202 Apr Cmr Tcr; carries ColE1 origin and mob site; unable to replicate in A. borkumensis 40 pSUP202::ABO_1185 Sm Fusion of pSUP202 and ABO_1185 Sm fragment of pCR2.1-TOPO via BamHI/HindIII sites; carries mob site; Cmr Apr Smr This study a For abbreviations for genotypes of E. coli, see reference 7.

Techniques: Mutagenesis, Construct, Solvent, Staining

Journal: Applied and Environmental Microbiology

Article Title: Isolation and Characterization of a Mutant of the Marine Bacterium Alcanivorax borkumensis SK2 Defective in Lipid Biosynthesis

doi: 10.1128/aem.02832-09

Figure Lengend Snippet: FIG. 4. Profiles of biomass production and fatty acid content and TLC analyses for the different A. borkumensis strains during cultivation with pyruvate as the sole carbon source. Cells were cultivated aerobi- cally in ONR7a medium containing 1% (wt/vol) sodium pyruvate at 30°C and 150 rpm. (A) Samples were taken every 12 h and assayed for biomass and TAG content as described in Materials and Methods. Solid lines represent the growth curves, and dotted lines represent the fatty acid contents. SK2, A. borkumensis wild type; atfA1 atfA2, A. borkumensis atfA1Km atfA2Sm; atfA1, A. borkumensis atfA1Km; atfA2, A. borkumensis atfA2Sm; 2 M131, transposon-induced mutant. (B) After 72 h of cultivation, cells were harvested, washed, and lyoph- ilized and the lipids were extracted with chloroform-methanol (2:1, vol/vol). Lipid extracts obtained from 5 mg lyophilized cells were ap- plied in each lane. The solvent system used for elution was hexane- diethyl ether-acetic acid (80:20:1, vol/vol/vol), and lipids were visual- ized by staining with iodine vapor. Lanes: T, triolein; OA, oleic acid; OO, oleyl oleate; SK2, A. borkumensis wild type; atfA1, A. borkumensis atfA1Km; atfA2, A. borkumensis atfA2Sm; atfA1 atfA2, A. borku- mensis atfA1Km atfA2Sm; and 2 M131, transposon-induced mutant. FA, fatty acids.

Article Snippet: Bacterial strains and plasmids used in this study Strain or plasmid Relevant characteristicsa Source and/orreference A. borkumensis strains SK2 (DSM 11573) Type strain; wild type; TAG and WE producer 49; DSMZ atfA1 Km mutant atfA1 disruption mutant; Kmr 28 atfA2 Sm mutant atfA2 disruption mutant; Smr 28 atfA1 Km atfA2 Sm double mutant atfA1 atfA2 double disruption mutant; Kmr Smr 28 ABO_1185 Sm mutant ABO_1185 disruption mutant; Smr; derivative of SK2 This study E. coli strains SM10 ( pir) Apr Cmr Kmr pUTmini-Tn5Cm 13 TOP10 F mcrA (mrr-hsdRMS-mcrBC) rpcL nupG endA1 deoR 80lacZ M15 lacX74 recA1 araD139 (ara-leu)7697 galU galK Invitrogen S17-1 recA1 thi-1 hsdR17(rK mK ) proA tra-RP4 40 Plasmids pBluescript SK( ) Plasmid used for subcloning of DNA; Apr lacPOZ Stratagene pUTmini-Tn5Cm Suicide vector for mutagenesis with mini-Tn5; Cmr 13 pCR2.1-TOPO Plasmid used for subcloning of DNA; Apr Invitrogen pCR2.1-TOPO::ABO_1185 Derivative of pCR2.1-TOPO containing 1,908-bp ABO_1185 gene This study pCR2.1-TOPO::ABO_1185 Sm Sm cassette cloned into NcoI site of pCR2.1-TOPO::ABO_1185; Apr Smr This study pSUP202 Apr Cmr Tcr; carries ColE1 origin and mob site; unable to replicate in A. borkumensis 40 pSUP202::ABO_1185 Sm Fusion of pSUP202 and ABO_1185 Sm fragment of pCR2.1-TOPO via BamHI/HindIII sites; carries mob site; Cmr Apr Smr This study a For abbreviations for genotypes of E. coli, see reference 7.

Techniques: Mutagenesis, Solvent, Staining

Journal: Applied and Environmental Microbiology

Article Title: Isolation and Characterization of a Mutant of the Marine Bacterium Alcanivorax borkumensis SK2 Defective in Lipid Biosynthesis

doi: 10.1128/aem.02832-09

Figure Lengend Snippet: FIG. 5. Mobilization of stored TAGs by A. borkumensis strains as revealed by TLC analysis. Cells were cultivated in ONR7a medium containing 1% (wt/vol) sodium pyruvate for 72 h at 30°C to promote accumulation of TAGs. Afterwards, cells were harvested, washed twice, and resuspended in carbon-free ONR7a medium to permit the mobilization of TAGs. Samples were taken at the beginning of culti- vation (time zero [T0]) and after 24 h (time point 1 [T1]), 48 h (T2),

Article Snippet: Bacterial strains and plasmids used in this study Strain or plasmid Relevant characteristicsa Source and/orreference A. borkumensis strains SK2 (DSM 11573) Type strain; wild type; TAG and WE producer 49; DSMZ atfA1 Km mutant atfA1 disruption mutant; Kmr 28 atfA2 Sm mutant atfA2 disruption mutant; Smr 28 atfA1 Km atfA2 Sm double mutant atfA1 atfA2 double disruption mutant; Kmr Smr 28 ABO_1185 Sm mutant ABO_1185 disruption mutant; Smr; derivative of SK2 This study E. coli strains SM10 ( pir) Apr Cmr Kmr pUTmini-Tn5Cm 13 TOP10 F mcrA (mrr-hsdRMS-mcrBC) rpcL nupG endA1 deoR 80lacZ M15 lacX74 recA1 araD139 (ara-leu)7697 galU galK Invitrogen S17-1 recA1 thi-1 hsdR17(rK mK ) proA tra-RP4 40 Plasmids pBluescript SK( ) Plasmid used for subcloning of DNA; Apr lacPOZ Stratagene pUTmini-Tn5Cm Suicide vector for mutagenesis with mini-Tn5; Cmr 13 pCR2.1-TOPO Plasmid used for subcloning of DNA; Apr Invitrogen pCR2.1-TOPO::ABO_1185 Derivative of pCR2.1-TOPO containing 1,908-bp ABO_1185 gene This study pCR2.1-TOPO::ABO_1185 Sm Sm cassette cloned into NcoI site of pCR2.1-TOPO::ABO_1185; Apr Smr This study pSUP202 Apr Cmr Tcr; carries ColE1 origin and mob site; unable to replicate in A. borkumensis 40 pSUP202::ABO_1185 Sm Fusion of pSUP202 and ABO_1185 Sm fragment of pCR2.1-TOPO via BamHI/HindIII sites; carries mob site; Cmr Apr Smr This study a For abbreviations for genotypes of E. coli, see reference 7.

Techniques:

Journal: Applied and Environmental Microbiology

Article Title: Isolation and Characterization of a Mutant of the Marine Bacterium Alcanivorax borkumensis SK2 Defective in Lipid Biosynthesis

doi: 10.1128/aem.02832-09

Figure Lengend Snippet: FIG. 6. Survival of A. borkumensis strains in carbon-free medium during starvation experiments. Viable cell counts (expressed in CFU) over the time course of carbon starvation were obtained as described in Materials and Methods. Values are means errors of results from triplicate determinations.

Article Snippet: Bacterial strains and plasmids used in this study Strain or plasmid Relevant characteristicsa Source and/orreference A. borkumensis strains SK2 (DSM 11573) Type strain; wild type; TAG and WE producer 49; DSMZ atfA1 Km mutant atfA1 disruption mutant; Kmr 28 atfA2 Sm mutant atfA2 disruption mutant; Smr 28 atfA1 Km atfA2 Sm double mutant atfA1 atfA2 double disruption mutant; Kmr Smr 28 ABO_1185 Sm mutant ABO_1185 disruption mutant; Smr; derivative of SK2 This study E. coli strains SM10 ( pir) Apr Cmr Kmr pUTmini-Tn5Cm 13 TOP10 F mcrA (mrr-hsdRMS-mcrBC) rpcL nupG endA1 deoR 80lacZ M15 lacX74 recA1 araD139 (ara-leu)7697 galU galK Invitrogen S17-1 recA1 thi-1 hsdR17(rK mK ) proA tra-RP4 40 Plasmids pBluescript SK( ) Plasmid used for subcloning of DNA; Apr lacPOZ Stratagene pUTmini-Tn5Cm Suicide vector for mutagenesis with mini-Tn5; Cmr 13 pCR2.1-TOPO Plasmid used for subcloning of DNA; Apr Invitrogen pCR2.1-TOPO::ABO_1185 Derivative of pCR2.1-TOPO containing 1,908-bp ABO_1185 gene This study pCR2.1-TOPO::ABO_1185 Sm Sm cassette cloned into NcoI site of pCR2.1-TOPO::ABO_1185; Apr Smr This study pSUP202 Apr Cmr Tcr; carries ColE1 origin and mob site; unable to replicate in A. borkumensis 40 pSUP202::ABO_1185 Sm Fusion of pSUP202 and ABO_1185 Sm fragment of pCR2.1-TOPO via BamHI/HindIII sites; carries mob site; Cmr Apr Smr This study a For abbreviations for genotypes of E. coli, see reference 7.

Techniques:

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: PLoS Pathogens

Article Title: Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection

doi: 10.1371/journal.ppat.1010794

Figure Lengend Snippet: ( A ) A549 cells were infected with influenza A/WSN/33 (H1N1) virus (WSN) or ( B ) influenza A/PR/8/34 (H1N1) virus (PR8) at an MOI of 0.5 for the indicated times. The mRNA levels of SPHK2 were analyzed by qRT-PCR at 0, 6, 12, 24 and 48 hpi, which was normalized to GAPDH. Error bars represent mean values ± SD calculated from the results for three individual samples. **, P<0.01; ***, P<0.001; ****, P<0.0001. ( C ) A549 cells were infected with influenza A/WSN/33 (H1N1) virus (WSN) or influenza A/PR/8/34 (H1N1) virus (PR8) at an MOI of 0.5 for 24 h. The protein level of SPHK2 was measured by Western blotting. NP, M1 and NS1 were used as positive controls of IAV infection, and β-actin was an internal loading control.

Article Snippet: After a vortex at 4°C for 1 h, lysed cells were centrifuged, the supernatants were obtained and incubated with the indicated antibodies (anti-Myc (M, M4439), anti-Flag (M, F1804) antibody from Sigma or anti-human SPHK2 (R, 17096-1-AP) antibody from Proteintech) and protein A/G affinity beads (Beyotime) at 4°C for 4 h with gentle shaking.

Techniques: Infection, Virus, Quantitative RT-PCR, Western Blot, Control

Journal: PLoS Pathogens

Article Title: Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection

doi: 10.1371/journal.ppat.1010794

Figure Lengend Snippet: A549 cells were uninfected or infected with influenza A/WSN/33 (H1N1) virus (WSN) at an MOI of 0.5 for 6 h. ( A ) Cells were fixed and stained using DAPI for nuclei (Blue), as well as anti-influenza NP (Green) and anti-SPHK2 antibodies (Red). The cells were visualized by confocal laser scanning microscopy. ( B ) The protein levels of SPHK2 and IAV NP in uninfected or infected A549 cells were detected by Western blotting, β-actin and Lamin B were used as loading controls for cytoplasmic and nuclear proteins, respectively. Results are representatives of three independent experiments.

Article Snippet: After a vortex at 4°C for 1 h, lysed cells were centrifuged, the supernatants were obtained and incubated with the indicated antibodies (anti-Myc (M, M4439), anti-Flag (M, F1804) antibody from Sigma or anti-human SPHK2 (R, 17096-1-AP) antibody from Proteintech) and protein A/G affinity beads (Beyotime) at 4°C for 4 h with gentle shaking.

Techniques: Infection, Virus, Staining, Confocal Laser Scanning Microscopy, Western Blot

Journal: PLoS Pathogens

Article Title: Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection

doi: 10.1371/journal.ppat.1010794

Figure Lengend Snippet: ( A ) A549 cells were transfected with plasmids encoding SPHK2 or a control vector pCMV-Myc, and 24 h after transfection, cells were infected with WSN virus at an MOI of 0.5. A549 cells or sgSPHK2-1 A549 cells were infected with WSN virus at an MOI of 0.5. At 24 hpi, Western blotting analysis was performed to detect viral NP, M1, NS1 and β-actin is shown as a loading control. ( B ) Virus titers in IAV (MOI = 0.5) infected A549 cells transfected with the plasmids pCMV-myc or Myc-SPHK2 and virus titers in IAV infected A549 cells or IAV infected sgSPHK2-1 A549 cells was determined in MDCK cells by plaque assay. Values are means ± SD (n = 3). Data are representative of three independent experiments. **, P<0.01; ****, P<0.0001.

Article Snippet: After a vortex at 4°C for 1 h, lysed cells were centrifuged, the supernatants were obtained and incubated with the indicated antibodies (anti-Myc (M, M4439), anti-Flag (M, F1804) antibody from Sigma or anti-human SPHK2 (R, 17096-1-AP) antibody from Proteintech) and protein A/G affinity beads (Beyotime) at 4°C for 4 h with gentle shaking.

Techniques: Transfection, Control, Plasmid Preparation, Infection, Virus, Western Blot, Plaque Assay

Journal: PLoS Pathogens

Article Title: Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection

doi: 10.1371/journal.ppat.1010794

Figure Lengend Snippet: ( A ) HDAC1 was stably knocked down in A549 cells. The mRNA and protein levels of HDAC1 was measured by qPCR and Western blotting respectively. ( B ) A549 cells were transfected with negative control (CTRL-siRNA) or SPHK2-targeting siRNAs (SPHK2-siRNAs) to silence SPHK2, and 48 h later, the mRNA and protein levels of SPHK2 was measured by qPCR and Western blotting respectively. ( C ) shHDAC1 A549 cells were transfected with siRNAs (CTRL-siRNA or SPHK2-siRNA-1, SPHK2-siRNA-2) and infected with WSN virus at an MOI of 0.5. After 24 h infection, the expression of NP, M1 and NS1 were detected by Western blotting, β-actin was as an internal loading control. ( D ) shCTRL A549 cells or shHDAC1 A549 cells were transfected with control vector or SPHK2-expressing plasmids and infected with WSN virus at an MOI of 0.5. After 24 h infection, the expression of NP, M1 and NS1 were detected by Western blotting. ( E ) shHDAC1 A549 cells were transfected with siRNAs (CTRL-siRNA or SPHK2-siRNA-1) and HDAC1 expression was restored. After 24 h, the cells were infected with WSN virus at an MOI of 0.5 and the expression of NP, M1 and NS1 were detected by Western blotting. ( F ) HDAC1 knock-down A549 cells (shHDAC1) were treated with CTRL-siRNA, SPHK2-siRNA-1 or transfected with Myc-SPHK2 plasmids, followed by restoration of HDAC1 expression. After 24 h, the cells were infected with WSN virus at an MOI of 0.5 and the virus titers were measured by plaque assay, and Myc-tagged HDAC1, SPHK2 protein in these cells were detected by Western blotting. Data are representative of three independent experiments. ns, no significance; **, P<0.01; ****, P<0.0001.

Article Snippet: After a vortex at 4°C for 1 h, lysed cells were centrifuged, the supernatants were obtained and incubated with the indicated antibodies (anti-Myc (M, M4439), anti-Flag (M, F1804) antibody from Sigma or anti-human SPHK2 (R, 17096-1-AP) antibody from Proteintech) and protein A/G affinity beads (Beyotime) at 4°C for 4 h with gentle shaking.

Techniques: Stable Transfection, Western Blot, Transfection, Negative Control, Infection, Virus, Expressing, Control, Plasmid Preparation, Knockdown, Plaque Assay

Journal: PLoS Pathogens

Article Title: Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection

doi: 10.1371/journal.ppat.1010794

Figure Lengend Snippet: ( A ) The amino acid sequence of SPHK2 wild-type (WT) and the mutation site of SPHK2-G212E are indicated. ( B and C ) A549 cells were transfected with vectors encoding Myc-tagged SPHK2-WT or SPHK2-G212E mutants, pCMV-Myc plasmid was as a control vector (-), and infected with WSN virus at an MOI of 0.5. After 24 h, Western blotting was used to detected the NP, M1 and NS1 protein (B). After 24 h infection, cell supernatants were collected for a plaque assay to measure the virus titers in A549 cells at each condition (C). All data are representative of three independent experiments showing similar results. ns, no significance; ***, P<0.001.

Article Snippet: After a vortex at 4°C for 1 h, lysed cells were centrifuged, the supernatants were obtained and incubated with the indicated antibodies (anti-Myc (M, M4439), anti-Flag (M, F1804) antibody from Sigma or anti-human SPHK2 (R, 17096-1-AP) antibody from Proteintech) and protein A/G affinity beads (Beyotime) at 4°C for 4 h with gentle shaking.

Techniques: Sequencing, Mutagenesis, Transfection, Plasmid Preparation, Control, Infection, Virus, Western Blot, Plaque Assay

Journal: PLoS Pathogens

Article Title: Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection

doi: 10.1371/journal.ppat.1010794

Figure Lengend Snippet: ( A and B ) HEK293 cells were transfected with pCMV-Myc (-) or SPHK2-encoding plasmids, with luciferase plasmids (pGL3-IFNβ-promoter or pGL3-NF-κB-promoter with pRL-TK co-transfected into cells), and then infected with WSN virus at an MOI of 0.5. NF-κB (A) or IFN-β (B) activity was measured using luciferase reporter assays, and viral NP, M1, NS1 proteins in these cells were detected by Western blotting, β-actin was as an internal loading control. ( C ) A549 cells or SPHK2 knockout A549 cells were infected with WSN virus at an MOI of 0.5. IFN-β activity was measured using luciferase reporter assays, and viral NP, M1, NS1 proteins in these cells were detected by Western blotting. ( D ) A549 cells were transfected with pCMV-Myc (-) or SPHK2-expressing plasmids, and 24 h after transfection, cell were infected with WSN virus at an MOI of 0.5. The mRNA levels of IFIT1, ISG15 and IFNb was analyzed by qPCR at 24 hpi, which was normalized to the mRNA level of GAPDH. ( E ) A549 cells or SPHK2 knockout A549 cells were infected with WSN virus at an MOI of 0.5 for 24 h. The mRNA levels of IFIT1, ISG15 and IFNb was analyzed by qPCR at 24 hpi. ( F ) A549 cells were transfected with pCMV-Myc or Myc-SPHK2 plasmids, and 24 h after transfection, cells were infected with WSN virus at an MOI of 0.5. pSTAT1, STAT1, NP, M1 and NS1 protein in A549 cells were analyzed by Western blotting at 24 hpi. ( G ) A549 cells or SPHK2 knockout A549 cells were infected with WSN virus at an MOI of 0.5 for 24 h. pSTAT1, STAT1, NP, M1 and NS1 protein in A549 cells were analyzed by Western blotting at 24 hpi. Data are representative of three independent experiments. ns, no significance; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Article Snippet: After a vortex at 4°C for 1 h, lysed cells were centrifuged, the supernatants were obtained and incubated with the indicated antibodies (anti-Myc (M, M4439), anti-Flag (M, F1804) antibody from Sigma or anti-human SPHK2 (R, 17096-1-AP) antibody from Proteintech) and protein A/G affinity beads (Beyotime) at 4°C for 4 h with gentle shaking.

Techniques: Transfection, Luciferase, Infection, Virus, Activity Assay, Western Blot, Control, Knock-Out, Expressing

Journal: PLoS Pathogens

Article Title: Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection

doi: 10.1371/journal.ppat.1010794

Figure Lengend Snippet: ( A ) A549 cells transfected with pCMV-Myc or Myc-SPHK2 were immunoprecipitated using anti-Myc antibody, and then Myc-tagged SPHK2, HDAC1, and TET3 proteins were analyzed by immunoblotting. ( B ) A549 cells transfected with pRK11-Flag or Flag-TET3 were immunoprecipitated using anti-Flag antibody, and then SPHK2, HDAC1, and Flag-tagged TET3 proteins were analyzed by immunoblotting. ( C ) A549 cells transfected with pCMV-Myc or Myc-HDAC1 were immunoprecipitated using anti-Myc antibody, and then SPHK2, Myc-tagged HDAC1, and TET3 proteins were analyzed by immunoblotting. ( D ) A549 cells uninfected or infected with WSN were immunoprecipitated using anti-Human SPHK2 polyclonal antibody (Proteintech, 17096-1-AP), and then SPHK2, HDAC1 and TET3 proteins were analyzed by immunoblotting. ( E ) Diagram of SPHK2-FL (full length) and deletion of SPHK2 fragments (F1, F2 and F3). ( F ) A 549 cells transfected with pCMV-Myc, Myc-SPHK2-FL, Myc-SPHK2-F1, Myc-SPHK2-F2, and Myc-SPHK2-F3 were immunoprecipitated by using anti-Myc antibody, and then Myc-tagged SPHK2, Myc-tagged SPHK2-F3, HDAC1, and TET3 proteins were analyzed by immunoblotting. ( G ) HEK293 cells were transfected with pCMV-Myc, Myc-SPHK2-FL, Myc-SPHK2-F1, Myc-SPHK2-F2, or Myc-SPHK2-F3, and then infected with WSN virus at an MOI of 0.5. IFN-β activity was measured by using luciferase reporter assays. ( H ) A549 cells transfected with pCMV-Myc (Mock), Myc-SPHK2 or Myc-SPHK2-F3, and IFN-β promoter enrichment for Myc-tagged SPHK2 or Myc-tagged SPHK2-F3 was detected by ChIP-qPCR. ( I ) IFN-β promoter enrichment for Myc-tagged HDAC1 in sgCTRL A549 cells or sgSPHK2-1 A549 cells overexpressed with Myc-tagged HDAC1 was detected by ChIP-qPCR. ( J ) A549 cells were transfected with CTRL-siRNA or TET3-siRNA, and 24 h post-transfection, cells were overexpressed with SPHK2. IFN-β promoter enrichment for Myc-tagged SPHK2 was detected by ChIP-qPCR. ( K ) A549 cells were uninfected or infected with WSN, and IFN-β promoter enrichment for SPHK2 was determined by ChIP-qPCR. Data are representative of three independent experiments. ns, no significance; **, P<0.01; ***, P<0.001.

Article Snippet: After a vortex at 4°C for 1 h, lysed cells were centrifuged, the supernatants were obtained and incubated with the indicated antibodies (anti-Myc (M, M4439), anti-Flag (M, F1804) antibody from Sigma or anti-human SPHK2 (R, 17096-1-AP) antibody from Proteintech) and protein A/G affinity beads (Beyotime) at 4°C for 4 h with gentle shaking.

Techniques: Transfection, Immunoprecipitation, Western Blot, Infection, Virus, Activity Assay, Luciferase, ChIP-qPCR

Journal: PLoS Pathogens

Article Title: Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection

doi: 10.1371/journal.ppat.1010794

Figure Lengend Snippet: ( A ) A549 cells and sgSPHK2-1 A549 cells were infected with IAV (WSN), MOI = 0.5, 24 h post infection, IFN-β promoter enrichment for H3ac, H4ac and HDAC1 antibodies was detected by ChIP-qPCR. ( B ) A549 cells transfected with pCMV-Myc or Myc-SPHK2 were infected with IAV (WSN), MOI = 0.5, 24 h post infection, IFN-β promoter enrichment for H3ac, H4ac and HDAC1 antibodies was detected by ChIP-qPCR. ( C ) A549 cells were treated with solvent (-) or TSA (5, 10, 20 or 50 nM) for 72 h, cell viability was detected by Cell Counting Kit-8. ( D ) A549 cells were treated with TSA (5, 10 and 20 nM), then transfected with Myc-SPHK2 and infected with WSN, MOI = 0.5, after 24 h, IFN-β mRNA level was measured by qPCR, and normalized to GAPDH. (E ) A549 cells were transfected with TET3-siRNA, then transfected with Myc-SPHK2 and infected with WSN, MOI = 0.5, after 24 h, cells IFN-β mRNA level was measured by qPCR. Data are representative of three independent experiments. ns, no significance; *, P<0.05; **, P<0.01; ***, P<0.001.

Article Snippet: After a vortex at 4°C for 1 h, lysed cells were centrifuged, the supernatants were obtained and incubated with the indicated antibodies (anti-Myc (M, M4439), anti-Flag (M, F1804) antibody from Sigma or anti-human SPHK2 (R, 17096-1-AP) antibody from Proteintech) and protein A/G affinity beads (Beyotime) at 4°C for 4 h with gentle shaking.

Techniques: Infection, ChIP-qPCR, Transfection, Solvent, Cell Counting

Journal: PLoS Pathogens

Article Title: Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection

doi: 10.1371/journal.ppat.1010794

Figure Lengend Snippet: The expression of SPHK2 in IAV infected cells is upregulated and then the location of SPHK2 is shifted from cytoplasm to nucleus. Afterwards, SPHK2 accumulated in the nucleus interacts with HDAC1 and its substrate binding domain also interacts with TET3 that bands to IFN-β promoter. Therefore, HDAC1 is recruited to IFN-β promoter and inhibited IFN-β transcription by enhancing the deacetylation of IFN-β promoter.

Article Snippet: After a vortex at 4°C for 1 h, lysed cells were centrifuged, the supernatants were obtained and incubated with the indicated antibodies (anti-Myc (M, M4439), anti-Flag (M, F1804) antibody from Sigma or anti-human SPHK2 (R, 17096-1-AP) antibody from Proteintech) and protein A/G affinity beads (Beyotime) at 4°C for 4 h with gentle shaking.

Techniques: Expressing, Infection, Binding Assay

Journal: PLoS Pathogens

Article Title: Enzymatic independent role of sphingosine kinase 2 in regulating the expression of type I interferon during influenza A virus infection

doi: 10.1371/journal.ppat.1010794

Figure Lengend Snippet: Plasmid constructs used in the study.

Article Snippet: After a vortex at 4°C for 1 h, lysed cells were centrifuged, the supernatants were obtained and incubated with the indicated antibodies (anti-Myc (M, M4439), anti-Flag (M, F1804) antibody from Sigma or anti-human SPHK2 (R, 17096-1-AP) antibody from Proteintech) and protein A/G affinity beads (Beyotime) at 4°C for 4 h with gentle shaking.

Techniques: Plasmid Preparation, Construct, FLAG-tag, Sequencing, Mutagenesis

Journal: BoneKEy Reports

Article Title: TGFβ-Mediated induction of SphK1 as a potential determinant in human MDA-MB-231 breast cancer cell bone metastasis

doi: 10.1038/bonekey.2015.88

Figure Lengend Snippet: Query of sphingolipid metabolism and S1P signaling receptor genes from low, median and highly metastatic MDA-MB-231 sublines. Previously described microarray data obtained from various MDA-MB-231 sublines of varying metastatic capacity were queried for predictive value to the expression of genes involved in sphingolipid/S1P metabolism and signaling. Technical details of these studies have been described previously.24 (a) Displays a simplified schematic of sphingolipid/S1P metabolism and S1PR receptor genes (italicized) chosen for this retrospective analysis. TGFβ1-induced SPHK1 expression was identified and provided significant predictive value between low, median and high metastatic behavior of the various MDA-MB-231 (SCP-derived) sublines in mouse metastasis models (b). **P<0.01 (Student's t-test, low vs median/high).

Article Snippet: Approximately 150 μg of cell extract (∼1 ml) was used per IP using a 1:1 combination (5 μg total) of two SphK1 antibodies: anti-SphK1 (Cat# AP7237c; Abgent Inc.) and anti-SphK1 (Cat# SP1621; ECM Biosciences, Versailles, KY, USA).

Techniques: Microarray, Expressing, Derivative Assay

Journal: BoneKEy Reports

Article Title: TGFβ-Mediated induction of SphK1 as a potential determinant in human MDA-MB-231 breast cancer cell bone metastasis

doi: 10.1038/bonekey.2015.88

Figure Lengend Snippet: qPCR screen for TGFβ1-mediated regulation of sphingosine/S1P metabolizing enzymes in human cancer cell lines. Human cancer cell lines grown in monolayer cultures were treated with 5 ng ml−1 hTGFβ1 for 2, 8 or 24 h followed by RNA isolation and Taqman-based qRT-PCR of SPHK1, sphingosine kinase-1; SPHK2, sphingosine kinase-2; SGPP1, sphingosine 1-phosphate phosphatase -1; SGPP2, sphingosine 1-phosphate phosphatase -2; and SGPL1, sphingosine 1-phosphate lyase. All mRNA values were normalized to GAPDH mRNA and plotted as fold induction versus the untreated control group (value=1). Only detectable/measurable mRNAs for each gene are displayed on their respective graphs.

Article Snippet: Approximately 150 μg of cell extract (∼1 ml) was used per IP using a 1:1 combination (5 μg total) of two SphK1 antibodies: anti-SphK1 (Cat# AP7237c; Abgent Inc.) and anti-SphK1 (Cat# SP1621; ECM Biosciences, Versailles, KY, USA).

Techniques: Isolation, Quantitative RT-PCR

Journal: BoneKEy Reports

Article Title: TGFβ-Mediated induction of SphK1 as a potential determinant in human MDA-MB-231 breast cancer cell bone metastasis

doi: 10.1038/bonekey.2015.88

Figure Lengend Snippet: Effects of various TGFβ isoforms/family members and kinetics of SPHK1 expression in MDA-MB-231 cells. MDA-MB-231 cells were treated with recombinant hTGFβ1 (5 ng ml−1), hTGFβ2 (5 ng ml−1), hTGFβ3 (5 ng ml−1), hActivin-A (50 ng ml−1) or hBMP-2 (50 ng ml−1) for 8 h followed by qRT-PCR analysis (a). Cells treated with hypoxia mimetics L-mimosine (L-MIM) and dimethyloxalyl glycine (DMOG) were harvested after 24 h (a). Both time and dose response kinetic experiments for SPHK1 and PMEPA1 were treated for approximately 8 h followed by qRT-PCR analysis (b). All data were normalized to GAPDH mRNA, analyzed using the Δ/Δ CT method and plotted as fold induction versus the untreated control group (value=1x). All data are plotted as mean fold-induction±s.e.m., n=3 per treatment group. *P<0.05 and **P<0.01 versus control group (one-way ANOVA, Bonferroni-corrected).

Article Snippet: Approximately 150 μg of cell extract (∼1 ml) was used per IP using a 1:1 combination (5 μg total) of two SphK1 antibodies: anti-SphK1 (Cat# AP7237c; Abgent Inc.) and anti-SphK1 (Cat# SP1621; ECM Biosciences, Versailles, KY, USA).

Techniques: Expressing, Recombinant, Quantitative RT-PCR

Journal: BoneKEy Reports

Article Title: TGFβ-Mediated induction of SphK1 as a potential determinant in human MDA-MB-231 breast cancer cell bone metastasis

doi: 10.1038/bonekey.2015.88

Figure Lengend Snippet: TβR1/Alk5 and RNA Polymerase II dependency for TGFβ1-mediated induction of SPHK1 expression. MDA-MB-231 cells were pre-treated for 30 min with either 10 μM SD-208 (TβR1/Alk5 inhibitor), 5 μg ml−1 actinomycin D (RNA Pol-II inhibitor) or 1 μM cycloheximide (protein synthesis inhibitor) followed by continued treatment with each agent in combination with 5 ng ml−1 TGFβ1 for 8 h. Cells were harvested, RNA was isolated and qRT-PCR analysis of SPHK1 and PMEPA1 expression was performed. All data were normalized to GAPDH mRNA, analyzed using the Δ/Δ CT method and plotted as fold induction versus their respective control groups (value=1x). All data are plotted as mean fold induction±s.e.m., n=3 per treatment group. *P<0.05 and **P<0.01 versus control group (one-way ANOVA, Bonferroni-corrected).

Article Snippet: Approximately 150 μg of cell extract (∼1 ml) was used per IP using a 1:1 combination (5 μg total) of two SphK1 antibodies: anti-SphK1 (Cat# AP7237c; Abgent Inc.) and anti-SphK1 (Cat# SP1621; ECM Biosciences, Versailles, KY, USA).

Techniques: Expressing, Isolation, Quantitative RT-PCR

Journal: BoneKEy Reports

Article Title: TGFβ-Mediated induction of SphK1 as a potential determinant in human MDA-MB-231 breast cancer cell bone metastasis

doi: 10.1038/bonekey.2015.88

Figure Lengend Snippet: TGFβ1-mediated increase in SphK1 protein and kinase activity in MDA-MB-231 cells. MDA-MB-231 cells were treated for 2, 8 or 24 h with hTGFβ1 (5 ng ml−1). After TGFβ1 treatment, cells were lysed and protein separated via SDS-PAGE and western blotted for SphK1 and α-tubulin. For kinase assays, cell lysate was prepared and used for immunoprecipitation of SphK1 using a fixed combination of specific SphK1 (Abgent/ECM) antibodies. SphK1 kinase activity was assessed by the Sphingosine Kinase Activity Assay Kit (Echelon) in the absence or presence of the substrate sphingosine (600 μM). Sphk1 kinase activity was plotted as a bar graph function of ATP depletion using values interpolated from the ATP standard curve. All data are plotted as percent activity±s.e.m., n=3 per treatment group. *P<0.05 and **P<0.01 versus comparator group (one-way ANOVA, Tukey's HSD). Cellular sphingosine and S1P content were quantified via LC-MS/MS.

Article Snippet: Approximately 150 μg of cell extract (∼1 ml) was used per IP using a 1:1 combination (5 μg total) of two SphK1 antibodies: anti-SphK1 (Cat# AP7237c; Abgent Inc.) and anti-SphK1 (Cat# SP1621; ECM Biosciences, Versailles, KY, USA).

Techniques: Activity Assay, SDS Page, Western Blot, Immunoprecipitation, Kinase Assay, Liquid Chromatography with Mass Spectroscopy

Journal: BoneKEy Reports

Article Title: TGFβ-Mediated induction of SphK1 as a potential determinant in human MDA-MB-231 breast cancer cell bone metastasis

doi: 10.1038/bonekey.2015.88

Figure Lengend Snippet: ZNF nuclease-mediated targeting of SPHK1 in MDA-MB-231 and cytotoxic sensitivity to SphK inhibitors. MDA-MB-231 cells were transiently-transfected with a custom SphK1 ZNF nuclease genomic targeting cassette along with CMV–eGFP expressing plasmid followed by FACS-mediated cell sorting of cell pools of varying fluorescence intensity (P1, P2 and P3) and subsequent culture-expanded pools (P2.e and P3.e). Genomic PCR was performed to assess genomic modification of the SPHK1 locus in various transfected and expanded MDA-MB-231 cell pools. Positive genomic alteration is noted by a black arrow corresponding to a PCR products band at ∼150–160 nt by gel electrophoresis (a). To assess MDA-MB-231 cell viability, cells were seeded and treated with varying concentrations of DMS, SKI II, FTY720 and SEW2781 for 7 days followed by cell viability determination by Cell Titer Glo analysis. Table IC50 calculations were based on the concentration response values analyzed by four-parameter non-linear regression curve fitting using GraphPad Prism (ver6.0) from two independent studies (b).

Article Snippet: Approximately 150 μg of cell extract (∼1 ml) was used per IP using a 1:1 combination (5 μg total) of two SphK1 antibodies: anti-SphK1 (Cat# AP7237c; Abgent Inc.) and anti-SphK1 (Cat# SP1621; ECM Biosciences, Versailles, KY, USA).

Techniques: Transfection, Expressing, Plasmid Preparation, FACS, Fluorescence, Modification, Nucleic Acid Electrophoresis, Concentration Assay

Journal: BoneKEy Reports

Article Title: TGFβ-Mediated induction of SphK1 as a potential determinant in human MDA-MB-231 breast cancer cell bone metastasis

doi: 10.1038/bonekey.2015.88

Figure Lengend Snippet: Evaluation of the SPHK1 gene expression signature in human breast cancer. Agilent microarray data compiled as part of The Cancer Genome Atlas (TCGA) Project were queried for correlative gene co-expression profiles to previously identified TGFβ/Activin signaling, PAM50 and triple-negative breast cancer (TBNC) gene panels and molecular signatures. Analysis was performed using the cBioPortal (http://cbioportal.org) gateway and repository for cancer genomics data—specifically utilizing the TCGA (Nature) invasive breast cancer patient data.32,33,34,35,37

Article Snippet: Approximately 150 μg of cell extract (∼1 ml) was used per IP using a 1:1 combination (5 μg total) of two SphK1 antibodies: anti-SphK1 (Cat# AP7237c; Abgent Inc.) and anti-SphK1 (Cat# SP1621; ECM Biosciences, Versailles, KY, USA).

Techniques: Expressing, Microarray

Journal: Endocrinology

Article Title: Sphingosine kinase as a regulator of calcium entry through autocrine sphingosine 1-phosphate signaling in thyroid FRTL-5 cells.

doi: 10.1210/en.2009-0288

Figure Lengend Snippet: FIG. 6. Calyculin A-evoked calcium entry in cells transduced with wild- type SK1, the G82D dominant-negative SK1 mutant, or the wild-type SK2. A, Cells transduced with wild-type SK1 (trace a) or the dominant- negative mutant G82D of SK1 (trace c) were pretreated with 100 nM calyculin A, and 1 mM calcium was added. Trace b shows calyculin A- evoked calcium entry in mock-transduced cells. Each trace is representative of at least seven separate determinations. B, Summary of several experiments performed as described in A. Each bar gives the mean SEM of seven to 14 determinations. *, P 0.05. C, G82D cells were treated with 100 nM calyculin A, and 1 mM calcium was added (trace a). In trace b, 100 nM S1P was added to calyculin A-treated cells before the start of the measurement. Trace c indicates calyculin- evoked calcium entry in cells transduced with wild-type SK1. D, Summary of several experiments performed as described in C. The bar denoted G82DS1P indicates the restoration of calcium entry in calyculin A-treated G82D cells after addition of S1P, and wt indicates the calcium entry in wild-type transduced cells treated with calyculin before addition of calcium. Each bar gives the mean SEM of five to eight determinations. *, P 0.05 compared with wt; §, P 0.05 compared with G82D. E, Cells transfected with plasmid only (trace a) or wild-type SK2 (trace b) were treated with calyculin A, and 1 mM calcium was added. The traces are representative of six separate experiments. F, Simplified scheme for how Sph, S1P, and SK1 may regulate calcium entry in FRTL-5 cells, and the role of calyculin A (Caly A). PPA denotes a presently uncharacterized phosphatase. We cannot exclude the possibility that calyculin also blocks a phosphatase directly regulating the calcium entry pathway.

Article Snippet: Transient transfection of FRTL-5 cells with SK2 The expression vector pCMV6-XL4-SphK2 was bought from Origene (Rockville, MD).

Techniques: Transduction, Dominant Negative Mutation, Mutagenesis, Transfection, Plasmid Preparation