Journal: Human Molecular Genetics

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

doi: 10.1093/hmg/ddx046

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

Article Snippet: Etoposide was from Selleckchem (#S1225). pGW1-Htt ex1 -Q 17 -GFP and pGW1-Htt ex1 -Q 72 -GFP were described ( 18 , 19 ). pGW1-SK2-GFP and pGW1-SK2-mApple-NLS were cloned from pCMV6-XL4-SPHK2 (#SC113181, OriGene). pGW1-mHtt ex1 -Q 46 was derived from pGW1-mHtt ex1 -Q 46 -GFP plasmid ( 17 ).

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

Journal: Human Molecular Genetics

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

doi: 10.1093/hmg/ddx046

Figure Lengend Snippet: SK2 is neurotoxic to cortical and striatal neurons in a dose-dependent manner. (A) An example of survival analysis. Primary cortical neurons were transfected with GFP (a morphology and viability marker) and tracked with an automated microscope. Images collected after 24 h demonstrate the ability to return to the same field of neurons and to follow them over time. Each image is a montage of non-overlapping images captured in one well of a 24-well plate. Scale bar is 200 μm. A region from the original images at different time points is zoomed in to demonstrate longitudinal single-cell tracking (right panels). Black arrows depict three neurons that degenerate by 72 h (neurons 1 and 3) and by 96 h (neuron 2) after transfection. Neuron 4 survived the entire experiment. Scale bar is 50 μm. (B) Longitudinal imaging of a neuron expressing GFP and mApple (left panel) and a neuron expressing GFP and SK2-mApple-NLS (right panel). The left neuron remains alive throughout the experiment. The right neuron died by 72 h. Scale bar is 10 μm. (C) Primary cortical neurons were transfected either with GFP + mApple (control) or with GFP + SK2-mApple-NLS. Transfected neurons were tracked with an automated microscope. Cumulative risk of death was calculated from Kaplan–Meier curves (JMP software). Cumulative risk of death curves demonstrates that the SK2-mApple-NLS construct is neurotoxic. ***P < 0.001 (log-rank test). Two hundred neurons were analyzed. Results were pooled from three independent experiments. (D) Primary striatal neurons were transfected either with GFP + mApple (control) or with GFP + SK2-mApple-NLS. Transfected neurons were tracked with an automated microscope. Cumulative risk of death was calculated from Kaplan–Meier curves. Cumulative risk of death curves demonstrate that the SK2-mApple-NLS construct is also neurotoxic to striatal neurons. ***P < 0.001 (log-rank test). Two hundred neurons were analyzed. Results were pooled from three independent experiments. (E) The average mApple-fluorescence and SK2-mApple-NLS intensities and single neuron survival to determine the dose-dependent toxicity in cortical primary neurons that express the mApple or SK2-mApple-NLS construct. Neuronal survival is not affected by mApple expression. However, higher SK2-mApple-NLS intensity lead to higher risk of death. m(control) = 0.1487; m(SK2-mApple-NLS) = −0.4246. ***P < 0.0001 (t-test). One hundred fifty neurons were analyzed. Results were pooled from three independent experiments. (F) The average mApple-fluorescence and SK2-mApple-NLS fluorescence intensities and single neuron survival to determine the dose-dependent toxicity in striatal primary neurons that express the mApple or SK2-mApple-NLS construct. Note that neuronal survival is affected by SK2-mApple-NLS expression. m(control) = 0.1005; m(SK2-mApple-NLS) = -0.4476. ***P < 0.0001 (t-test). One hundred fifty neurons were analyzed. Results were pooled from three independent experiments.

Article Snippet: Etoposide was from Selleckchem (#S1225). pGW1-Htt ex1 -Q 17 -GFP and pGW1-Htt ex1 -Q 72 -GFP were described ( 18 , 19 ). pGW1-SK2-GFP and pGW1-SK2-mApple-NLS were cloned from pCMV6-XL4-SPHK2 (#SC113181, OriGene). pGW1-mHtt ex1 -Q 46 was derived from pGW1-mHtt ex1 -Q 46 -GFP plasmid ( 17 ).

Techniques: Transfection, Marker, Microscopy, Single Cell Tracking, Imaging, Expressing, Software, Construct, Fluorescence

Journal: Human Molecular Genetics

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

doi: 10.1093/hmg/ddx046

Figure Lengend Snippet: SK2 promotes formation of DNA DSBs in primary neurons. (A) Cortical neurons were transfected with mApple (control) or with SK2-mApple-NLS. At 24 h after transfection, cells were fixed, stained with an antibody against γH2A.X (a marker of DSBs) and with the nuclear Hoechst dye (DAPI), and imaged. As a positive control, neurons were treated with the DNA damaging drug etoposide. Note the γH2A.X puncta index in cells treated with etoposide and in SK2-mApple-NLS-expressing neurons is significantly greater than non-treated mApple-expressing neurons. Scale bar is 5 μm. See right panel for higher magnification image of γH2A.X. Scale bar is 2 μm. (B) Quantification of the γH2A.X puncta index in cortical and striatal neurons transfected with mApple (cont, control) or with SK2-mApple-NLS or transfected with mApple and treated with 5 µM etoposide (etop). ***P < 0.0001 (t-test). A.u., Arbitrary units. Three hundred neurons were analyzed from three independent experiments. (C) Cortical neurons were transfected with mApple or with SK2-mApple-NLS. At 24 h after transfection, cells were fixed, stained with an antibody against 53BP1 (a DNA damage repair protein) and with DAPI, and imaged. 53BP1 was diffuse in mApple-expressing neurons. In SK2-mApple-NLS-expressing neurons, 53BP1 formed puncta. Scale bar is 5 μm. See right panel for higher magnification image of 53BP1. Scale bar is 2 μm. (D) Quantification of the 53BP1 puncta index in cortical and striatal neurons transfected with mApple (cont) or with SK2-mApple-NLS. ***P = 0.0008 and ***P < 0.0001 (t-test) for cortical striatal neurons, respectively. A.u., arbitrary units. Three hundred neurons were analyzed from three independent experiments.

Article Snippet: Etoposide was from Selleckchem (#S1225). pGW1-Htt ex1 -Q 17 -GFP and pGW1-Htt ex1 -Q 72 -GFP were described ( 18 , 19 ). pGW1-SK2-GFP and pGW1-SK2-mApple-NLS were cloned from pCMV6-XL4-SPHK2 (#SC113181, OriGene). pGW1-mHtt ex1 -Q 46 was derived from pGW1-mHtt ex1 -Q 46 -GFP plasmid ( 17 ).

Techniques: Transfection, Staining, Marker, Positive Control, Expressing

Journal: Human Molecular Genetics

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

doi: 10.1093/hmg/ddx046

Figure Lengend Snippet: An inhibitor of SK2, ABC294640, mitigates neurotoxicity induced by ectopically expressed SK2. (A) Primary cortical neurons were treated with a vehicle or with different concentrations of ABC294640 (ABC, 1 and 5 µM). Neuronal lysates were then analyzed by western blotting with antibodies against phosphorylated SK2 (pSK2) and pan-SK2 (SK2). Actin was used as a loading control. (B) Quantification of pSK2 levels normalized to SK2 from (A). Results were pooled from three independent experiments. (C) Primary cortical neurons transfected with GFP and mApple (control) or with GFP and SK2-mApple-NLS constructs. Two cohorts of neurons that express SK2-mApple-NLS and GFP were treated with a vehicle (SK2-mApple-NLS) or 1 μM ABC294640 (SK2-mApple-NLS + ABC). Risk of death was calculated from Kaplan–Meier curves. The ABC294640 drug reduces the risk of death in SK2-mApple-NLS-expressing neurons. ***P < 0.001 (Log-Rank test). n.s., not significant (P = 0.06). (D) Primary striatal neurons transfected with GFP and mApple (control) or with GFP and SK2-mApple-NLS constructs. Neurons that express SK2-mApple-NLS and GFP were treated with a vehicle (SK2-mApple-NLS) or 1 μM ABC294640 (SK2-mApple-NLS + ABC). Risk of death was calculated from Kaplan–Meier curves. The ABC294640 drug reduces the risk of death in SK2-mApple-NLS-expressing striatal neurons. ***P < 0.001 (log-rank test), **P = 0.0058 (control vs SK2-mApple-NLS + ABC), and **P = 0.0061 (SK2-mApple-NLS vs SK2-mApple-NLS + ABC). One hundred fifty neurons were analyzed. Results were pooled from three independent experiments. (E) Cortical neurons were transfected with SK2-mApple-NLS, and treated with a vehicle (control) or 5 μM ABC294640 (ABC) for 24 h. Cells were fixed, stained with an antibody against γH2A.X and with the nuclear Hoechst dye (DAPI), and imaged. Note that SK2 inhibitor reduces the γH2A.X puncta index in SK2-mApple-NLS-expressing neurons compared to vehicle-treated transfected cells. Scale bar is 5 μm. (F) Quantification of the γH2A.X puncta index in cortical and striatal neurons transfected with SK2-mApple-NLS and treated with a vehicle (control) or 5 μM ABC294640 (ABC) for 24 h. ***P < 0.0001 (t-test). A.u., Arbitrary units. Three hundred neurons were analyzed from three independent experiments. (G) Quantification of the 53BP1 puncta index in cortical and striatal neurons transfected with SK2-mApple-NLS, treated with a vehicle (control) or 5 μM ABC294640 (ABC) for 24 h. **P = 0.0013 and **P = 0.0027 (t-test) for cortical and striatal neurons, respectively. A.u., Arbitrary units. Two hundred neurons were analyzed from three independent experiments. (H) Cortical neurons were transfected with SK2-mApple-NLS, and treated with a vehicle (control) or 5 μM ABC294640 (ABC) for 24 h. Cells were fixed, stained with an antibody against histone H4 (acetyl K5 + K8 + K12 + K16, acetyl-H4) and with the nuclear Hoechst dye (DAPI), and imaged. Scale bar is 5 μm. (I) Quantification of the acetyl-H4 puncta index in cortical and striatal neurons transfected with mApple (control) or with SK2-mApple-NLS construct. ***P = 0.0002 and ***P < 0.0001 (t-test) for cortical and striatal neurons, respectively. A.u., Arbitrary units. Two hundred neurons were analyzed from three independent experiments. (J) Quantification of the acetyl-H4 puncta index in cortical and striatal neurons transfected with SK2-mApple-NLS and treated with a vehicle (control) or with 5 μM ABC294640 (ABC). ***P = 0.003 and ***P = 0.0038 (t-test) for cortical and striatal neurons, respectively. A.u., Arbitrary units. Two hundred neurons were analyzed from three independent experiments.

Article Snippet: Etoposide was from Selleckchem (#S1225). pGW1-Htt ex1 -Q 17 -GFP and pGW1-Htt ex1 -Q 72 -GFP were described ( 18 , 19 ). pGW1-SK2-GFP and pGW1-SK2-mApple-NLS were cloned from pCMV6-XL4-SPHK2 (#SC113181, OriGene). pGW1-mHtt ex1 -Q 46 was derived from pGW1-mHtt ex1 -Q 46 -GFP plasmid ( 17 ).

Techniques: Western Blot, Transfection, Construct, Expressing, Staining

Journal: Human Molecular Genetics

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

doi: 10.1093/hmg/ddx046

Figure Lengend Snippet: SK2 is hyperphosphorylated in an HD mouse model. (A) The cortex and striatum were obtained from wild-type (WT) and BACHD (HD) mouse brains, homogenized and processed by immunoblotting with antibodies against mHtt. Actin was used as a loading control. (B) Cortices and striata were dissected from wild-type (WT) and BACHD (HD) mouse brains, homogenized, and processed by immunoblotting with antibodies against phosphorylated histone H2A.X, γH2A.X. Actin was used as a loading control. (C) Levels of γH2A.X were normalized to actin in WT and BACHD brain samples. *P = 0.018 and *P = 0.046 (t-test) for cortical and striatal neurons, respectively. Results were pooled from three WT and three BACHD mice. (D) The cortex and striatum were obtained from wild-type (WT) and BACHD (HD) mouse brains, homogenized and processed by immunoblotting with antibodies against pan-SK2 (SK2) and phosphorylated SK2 (pSK2). Actin was used as a loading control. (E) Levels of pan SK2 (pan) and phosphorylated SK2 (phospho) were normalized to actin in WT and BACHD brain samples. *P = 0.046 (t-test). n.s., not significant (P = 0.15). Results were pooled from three WT and three BACHD mice. (F) Cortical wild-type (WT) and BACHD (HD) neurons were transfected with mApple, fixed, and immunostained with an antibody against phosphorylated SK2 (pSK2), and with the nuclear Hoechst dye (DAPI). Scale bar is 10 μm. White arrows depict transfected neurons. (G) Quantification of the nuclear phosphorylated SK2 fluorescence intensity from (F). ***P < 0.001 (t-test). A.u., arbitrary units. Two hundred neurons were analyzed from two independent experiments.

Article Snippet: Etoposide was from Selleckchem (#S1225). pGW1-Htt ex1 -Q 17 -GFP and pGW1-Htt ex1 -Q 72 -GFP were described ( 18 , 19 ). pGW1-SK2-GFP and pGW1-SK2-mApple-NLS were cloned from pCMV6-XL4-SPHK2 (#SC113181, OriGene). pGW1-mHtt ex1 -Q 46 was derived from pGW1-mHtt ex1 -Q 46 -GFP plasmid ( 17 ).

Techniques: Western Blot, Transfection, Fluorescence

Journal: Human Molecular Genetics

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

doi: 10.1093/hmg/ddx046

Figure Lengend Snippet: ABC294640 mitigates mHtt-associated neurodegeneration in two primary neuron HD models. (A) Primary rat cortical neurons transfected with Httex1-Q17-GFP + mApple (a viability and morphology marker), or with mHttex1-Q72-GFP + mApple. Neurons transfected with mHttex1-Q72-GFP + mApple were treated with a vehicle or 1 μM ABC294640 (ABC), and tracked longitudinally. Exponential risk of death was calculated from Kaplan–Meier curves (JMP software). Risk of death curves demonstrate that ABC is neuroprotective and reduces the risk of death in mHttex1-Q72-GFP-expressing neurons. ***P < 0.001, and *P = 0.04 (log-rank test). One hundred fifty neurons were analyzed. Results were pooled from at least three independent experiments. (B) Primary striatal neurons transfected Httex1-Q17-GFP + mApple or with mHttex1-Q72-GFP + mApple. Neurons transfected with mHttex1-Q72-GFP + mApple were treated with a vehicle or 1 μM ABC294640 (ABC). Exponential risk of death was calculated from Kaplan–Meier curves. Risk of death curves show that ABC294640 is also neuroprotective for striatal neurons that express mHttex1. ***P < 0.001 (log-rank test). n.s., not significant (P = 0.1). One hundred fifty neurons were analyzed. Results were pooled from at least three independent experiments. (C) Primary cortical neurons were cultured from wild-type (WT) BACHD (HD) mouse pups, transfected with GFP (a viability and morphology marker), and treated with a vehicle or 1 μM ABC294640 (ABC). Exponential risk of death was calculated from Kaplan–Meier curves. Risk of death curves show that ABC reduces the risk of death in HD cortical neurons. ***P < 0.001, *P = 0.038 (log-rank test). Fifty neurons were analyzed from 3 WT mice and 3 HD mice. Results were pooled from two independent experiments. (D) Primary striatal neurons were cultured from wild-type (WT) and BACHD (HD) mouse pups, transfected with GFP (a viability and morphology marker), and treated with a vehicle or 1 μM ABC294640 (ABC). Exponential risk of death was calculated from Kaplan–Meier curves. Risk of death curves reveal that ABC is neuroprotective for HD striatal neurons. ***P < 0.001 (log-rank test). Fifty neurons were analyzed from 3 WT mice and 3 HD mice. Results were pooled from two independent experiments. (E) Cortical BACHD neurons were transfected with mApple (a viability and morphology marker) and scrambled siRNA (siControl) or with mApple and SK2-targeted siRNA (siSK2). Neurons were then fixed and immunostained with an antibody against SK2 and with the nuclear Hoechst dye. Scale bar is 10 μm. (F) Quantification of SK2 fluorescence intensity in neurons transfected with mApple and scrambled siRNA (siCont) or with mApple and SK2-targeted siRNA (siSK2). ***P < 0.001 (t-test). A.u., arbitrary units. Two hundred neurons were analyzed from two independent experiments. (G) Cortical and striatal neurons were transfected with mApple, mHttex1-Q46 and scrambled siRNA (siCont) or with mApple, mHttex1-Q46 and siRNA against SK2 mRNA (siSK2). Neurons were fixed 72 h after transfection and immunostained against γH2A.X. Neurons were imaged and analyzed. SK2 downregulation reduces γH2A.X puncta index, compared to controls. **P = 0.0036 and ***<0.0001 (t-test). A.u., arbitrary units. One hundred fifty neurons per condition were analyzed from two independent experiments. (H) Cortical BACHD neurons were transfected with mApple and scrambled siRNA (siControl) or with mApple and siRNA against SK2 mRNA (siSK2), and then tracked longitudinally. Risk of death was calculated from Kaplan–Meier curves. Risk of death curves demonstrates that reducing SK2 expression is neuroprotective and reduces the risk of death. ***P < 0.001 (log-rank test). (I) Striatal BACHD neurons were transfected with mApple and scrambled siRNA (siControl) or with mApple and siRNA against SK2 mRNA (siSK2), and then tracked longitudinally. Risk of death was calculated from Kaplan–Meier curves. Risk of death curves demonstrates that reducing SK2 expression is neuroprotective and reduces the risk of death in striatal cultures. ***P < 0.001 (log-rank test). Results were pooled from two independent experiments.

Article Snippet: Etoposide was from Selleckchem (#S1225). pGW1-Htt ex1 -Q 17 -GFP and pGW1-Htt ex1 -Q 72 -GFP were described ( 18 , 19 ). pGW1-SK2-GFP and pGW1-SK2-mApple-NLS were cloned from pCMV6-XL4-SPHK2 (#SC113181, OriGene). pGW1-mHtt ex1 -Q 46 was derived from pGW1-mHtt ex1 -Q 46 -GFP plasmid ( 17 ).

Techniques: Transfection, Marker, Software, Expressing, Cell Culture, Fluorescence

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