Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a ConSurf analysis of hSPNS2 protein. b Pairwise Alignment Scores for hSPNS2 protein and DNA sequences with putative homologs arranged according to the degree of sequence identity. c S1P levels in blood (n = 13, 15), lymph fluid (n = 10, 12), perfused liver, lung, kidney, heart (n = 3, 3). d Glucose levels in blood (n = 10,14), lymph fluid (n = 7, 13), perfused liver (n = 7, 5), lung (n = 7, 10), kidney (n = 5, 5), heart (n = 5, 5). e Glucose levels in urine (n = 7, 6), feces (n = 10, 8), tibialis anterior muscle (n = 5, 5), and gonadal adipose tissue (n = 4, 5). f Body weights (n = 20, 20). Body composition expressed as percentage of fat mass (n = 12, 12). g Food consumption, energy balance, energy expenditure, and locomotor activity (Beam breaks/h) determined with the PhenoMaster (n = 9,10; N = 3). h Blood hemoglobin concentration (n = 17, 9) and percentage of glycosylated hemoglobin, HbA1c (n = 7, 7). i Oral glucose tolerance test (GTT) and area under the curve (AUC) (n = 5, 5; N = 2). j Plasma levels of fasting insulin (n = 14, 14), glucagon (n = 8, 8) and thyroid hormone triiodothyronine (T3). Data are means ± s.e.m. k – n PET-CT imaging analysis of 2-FDG in Spns2 –/– mice. PET tracer 2-FDG (270–550 µCi) was gavaged prior to anesthetization of WT or Spns2 –/– mice for PET-CT scans. (n = 3, 3, N = 2). k , l At 60 min, tracer activity of target organs was quantified in volumes of interest (VOI). Data are percentage of whole-body activity for the right kidney (RK), heart, and bladder (Bl). l Coronal sections (0.4 mm thick) of PET images are presented according to a spectral scale for tracer activity, from red (highest), to green (intermediate), to blue (lowest). m The distribution of 2-FDG in representative WT at the specified times following gavage is shown to demonstrate the assessment of gastric emptying and intestinal absorption. St, stomach; In, intestines. n Representative images of the VOI in bladder to determine % of urinary excretion. o % of urinary excretion, gastric emptying and intestinal absorption. Data are means ± s.d. Two-tailed unpaired t-test. Source data are available for this figure in the Source Data file.

Article Snippet: To overexpress TurboGFP-tagged SPNS2 for ligand binding studies, SVEC4-10 cells were transfected with 2 μg of SPNS2 ( NM_001124758 ) Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene), 2 μg of Spns2 ( NM_153060 ) Mouse Tagged ORF Clone in pCMV6-AC-GFP (# MG218672 , Origene), or as controls with 2 μg of the pCMV6-AC-GFP vector (# PS100010 , Origene) or 2 μg of glucose transporter GLUT1 (SLC2A1) ( NM_006516 ), or Human Tagged ORF Clone in pCMV6-AC-GFP (#RG222696, Origene).

Techniques: Sequencing, Activity Assay, Concentration Assay, Clinical Proteomics, Positron Emission Tomography-Computed Tomography, Imaging, Two Tailed Test

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a Immunofluorescence localization of SPNS2 (red) on plasma membrane of SVEC4-10. b Glucose increases S1P secretion by SVEC4-10 cells (n = 3, N = 3). c SVEC4-10 cells were treated with S1P lyase inhibitor A6770 (200 µM) or with SPNS2 inhibitor SLF1081851 (2 µM) and levels of S1P in cells (n = 8–10, N = 5) and medium (n = 9, N = 3) as well as glucose uptake were determined (n = 7, N = 3). d SPNS2 expression in two SPNS2 stably overexpressing SVEC4-10 cell lines generated by CRISPR activation plasmids (CTL1, SPNS2-OE1) or lentiviral activation particles (CTL2, SPNS2-OE2) and in two SPNS2 deleted SVEC4-10 cell lines (SPNS2-KO1 generated with double nickase plasmids, and SPNS2-KO2 via CRISPR/Cas9 knock-out and homology-directed repair plasmids) compared to their controls. e – h S1P levels in cells and medium, and glucose uptake were measured in SPNS2-OE1 ( e ), SPNS2-OE2 ( f ), SPNS2-KO1 ( g ), SPNS2-KO2 ( h ). (n = 6, N = 3). i , j SPNS2-OE1, SPNS2-KO1 cells and their controls were treated with S1P (500 nM), and phosphorylation of p42/44 ( i ) and glucose uptake ( j ) were determined (n = 6, N = 3). k , l Glucose uptake in SPNS2-OE1, SPNS2-KO1 and their control cells treated with insulin (100 nM) or GLUT1 inhibitor BAY-876 (10 nM) (n = 3, N = 3). Data are means ± s.d. b One-way analysis of variance test followed by Šídák’s multiple comparisons test. c – l two-tailed unpaired t-test. Source data are available for this figure in the Source Data file.

Article Snippet: To overexpress TurboGFP-tagged SPNS2 for ligand binding studies, SVEC4-10 cells were transfected with 2 μg of SPNS2 ( NM_001124758 ) Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene), 2 μg of Spns2 ( NM_153060 ) Mouse Tagged ORF Clone in pCMV6-AC-GFP (# MG218672 , Origene), or as controls with 2 μg of the pCMV6-AC-GFP vector (# PS100010 , Origene) or 2 μg of glucose transporter GLUT1 (SLC2A1) ( NM_006516 ), or Human Tagged ORF Clone in pCMV6-AC-GFP (#RG222696, Origene).

Techniques: Immunofluorescence, Clinical Proteomics, Membrane, Expressing, Stable Transfection, Generated, CRISPR, Activation Assay, Knock-Out, Phospho-proteomics, Control, Two Tailed Test

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a Proliferation of Spns2 overexpressing (SPNS2-OE1) or Spns2 deleted cells (SPNS2-KO1) and controls cells. 10 5 cells were cultured in medium containing 4.5 g/L glucose without or with 500 nM S1P as indicated and cell numbers measured after 72 h (n = 3). b – f Cells were cultured in medium without or with 4.5 g/L glucose and/or 500 nM S1P as indicated. b , c Migration of cells in wound healing assays 24 h after creating a gap in a confluent monolayer and change to media containing glucose or S1P as indicated. b Representative images at 0 or 24 h after initiation of migration and ( c ) percentages of wound closures determined in cell migration assays in the presence of aphidicolin (n = 4). d – f ECIS measurements of the resistance of the indicated cells. d Representative continuous resistance measurements. e normalized endpoint resistance of the indicated cells cultured for 118 h. Resistance was normalized to the value measured at 12 h. f Normalized endpoint resistance of the indicated cells cultured for 118 h in the absence or presence of glucose and/or S1P (n = 3). g FITC-dextran leakage from the indicated cell monolayers cultured in the present of glucose (n = 3, 6). h FITC-dextran leakage from the indicated cell monolayers in the absence or presence of glucose and/or S1P (n = 4). a , c , e – h Data are means ± s.d. ns, not significant; One-way analysis of variance test followed by Sidak's multiple comparisons test or ( h ) Welch’s ANOVA multiple comparisons test. Source data are available for this figure in the Source Data file.

Article Snippet: To overexpress TurboGFP-tagged SPNS2 for ligand binding studies, SVEC4-10 cells were transfected with 2 μg of SPNS2 ( NM_001124758 ) Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene), 2 μg of Spns2 ( NM_153060 ) Mouse Tagged ORF Clone in pCMV6-AC-GFP (# MG218672 , Origene), or as controls with 2 μg of the pCMV6-AC-GFP vector (# PS100010 , Origene) or 2 μg of glucose transporter GLUT1 (SLC2A1) ( NM_006516 ), or Human Tagged ORF Clone in pCMV6-AC-GFP (#RG222696, Origene).

Techniques: Cell Culture, Migration

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a Molecular dynamics simulations of the inward-facing open conformation of hSPNS2 (PDB ID 8EX4) show that S1P can slither up as its hydrocarbon tail becomes vertically aligned from its kinked structure. b Changes in the vertical distance of the S1P phosphate headgroup from its original location near S232 of SPNS2. c The upward translocation of S1P causes noticeable structural changes at the extracellular vestibule of SPNS2. d The root-mean-squared deviation (RMSD) of the SPNS2 structure as S1P slithers upward during the unbiased simulation. The set of hydrophobic residues shown in Fig. 4a pose a barrier for the upward movement illustrated by the free-energy plot shown in Supplementary Fig. . Once the phosphate group makes it past these residues, further upward movement is relatively easier, and the hydrophobic residues close in to obstruct its downward movement. e – g Snapshots of the representative, inward-facing open structures of SPNS2-S1P complexes at various times during the MD simulation in which S1P moves upward. Transient glucose-binding pockets were obtained by molecular docking of glucose into 2000 frames extracted from the 1000 ns simulation. h – j Close-up views of the glucose-binding pockets are shown in ( e – g ). Residues near the most stable glucose-binding pockets are indicated. Lower panels highlight residues within 3.5 Å of glucose in the top (site 1), central (sites 2 and 3), and bottom (site 4) glucose-binding pockets and H-bonds are indicated with dotted lines. k Glucose molecule hydrogen bonds with S1P and nearby protein residues before transitioning of glucose to the intracellular side (during 150–300 ns in the distance plot shown in m ). l Trace the glucose pathway as it enters the intracellular side from the extracellular side. m Changes in the vertical distance of glucose from its initial position at the central site 3 binding pocket as it traverses through the channel and passes into the intracellular side. Source data are available for this figure in the Source Data file.

Article Snippet: To overexpress TurboGFP-tagged SPNS2 for ligand binding studies, SVEC4-10 cells were transfected with 2 μg of SPNS2 ( NM_001124758 ) Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene), 2 μg of Spns2 ( NM_153060 ) Mouse Tagged ORF Clone in pCMV6-AC-GFP (# MG218672 , Origene), or as controls with 2 μg of the pCMV6-AC-GFP vector (# PS100010 , Origene) or 2 μg of glucose transporter GLUT1 (SLC2A1) ( NM_006516 ), or Human Tagged ORF Clone in pCMV6-AC-GFP (#RG222696, Origene).

Techniques: Translocation Assay, Binding Assay

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a Representative image showing localization of SPNS2-TurboGFP and GLUT1-TurboGFP. b Temperature shift melting curves for purified hSPNS2-TurboGFP in the absence (blue) and presence of glucose (black). (n = 3, N = 3). Values are means ± s.d. c Apparent melting temperatures (T m ) for hSPNS2-TurboGFP, purified untagged hSPNS2 and purified mSPNS2-FLAG were calculated from the inflection points of the fitting curves. (n = 3, N = 3). Values are means ± s.e.m. Two-tailed unpaired t-test. d Schematic representation of the scintillation proximity assay (SPA). TurboGFP-tagged transport proteins are bound to scintillation beads. When radiolabeled glucose binds to these bead-bound transport proteins, emitted β-rays are close enough to stimulate the scintillation beads to emit light. Illustration created by Luciana Giono. e SPA signals of [ 3 H]glucose (0.8 μCi) binding to TurboGFP (vector), GLUT1-TurboGFP mSPNS2-TurboGFP, and hSPNS2-TurboGFP and its mutants E433A and T329A immobilized on the surfaces of polyvinyl toluene protein A-coated scintillation beads (500 μg per well) were measured by a scintillation counter. Values are means ± s.e.m. (n = 7; N = 2) and (n = 3; N = 3). One-way analysis of variance test followed by Dunnett’s multiple comparisons test. Source data are available for this figure in the Source Data file.

Article Snippet: To overexpress TurboGFP-tagged SPNS2 for ligand binding studies, SVEC4-10 cells were transfected with 2 μg of SPNS2 ( NM_001124758 ) Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene), 2 μg of Spns2 ( NM_153060 ) Mouse Tagged ORF Clone in pCMV6-AC-GFP (# MG218672 , Origene), or as controls with 2 μg of the pCMV6-AC-GFP vector (# PS100010 , Origene) or 2 μg of glucose transporter GLUT1 (SLC2A1) ( NM_006516 ), or Human Tagged ORF Clone in pCMV6-AC-GFP (#RG222696, Origene).

Techniques: Purification, Two Tailed Test, Scintillation Proximity Assay, Binding Assay, Plasmid Preparation

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a , b Identification of key SPNS2 residues involved in glucose transport. a Western blots and representative images of localization of hSPNS2-TurboGFP and its mutants. b Glucose uptake activities of SPNS2 variants with mutations in potential key residues involved in glucose or S1P engagement. hSPN S2 , GLUT1 vector, or the indicated mutants were overexpressed in SPNS2-KO1 cells lacking endogenous SPNS2. Glucose uptake was normalized to SPNS2 expression determined by GFP fluorescence ( n = 5, N = 3). Data are means ± s.e.m. One-way analysis of variance test followed by Dunnett’s multiple comparison test. c – f Direct glucose and S1P transport by SPNS2 proteoliposomes. c Illustration of cell-free preparation of SPNS2 proteoliposomes for functional transport analysis. d Comparable levels of hSPNS2 and its variants by western blots. e , f Proteoliposomes of WT hSPNS2 and variants were loaded without or with glucose ( e ) or S1P ( f ) as indicated and uptake of 1 μM NBD-S1P ( e ) or 1 µM NBD-glucose ( f ) determined. Arbitrary units (a.u.) (n = 3, N = 3). Nonspecific uptake was measured using protein-free liposomes (empty), vector containing proteoliposomes (vector), and T1R1 containing proteoliposomes (control). (n = 3, N = 3). One-way analysis of variance test followed by Dunnett’s multiple comparisons test. g Illustration of SPNS2-mediated export of S1P out of cells while transporting glucose inward. Illustrations in panels c and g created by Luciana Giono. h – j SPNS2-mediated D-[3H]glucose uptake. h Time-dependent specific uptake of D-[ 3 H]glucose into hSPNS2-containing proteoliposomes that were loaded without or with S1P (n = 3–5, N = 3). Nonspecific uptake measured using protein-free liposomes (empty) was subtracted from the specific uptake. i Uptake of [ 3 H]glucose by hSPNS2 or empty liposomes at 40 sec (n = 4, N = 3). j Kinetics of D-glucose uptake by hSPNS2. Specific uptake measured at 40 sec was calculated by subtraction of nonspecific [ 3 H]glucose uptake by empty liposomes and fitted to a non-linear regression analysis using Michaelis–Menten enzyme kinetics plot with K M , V max , and k cat values calculated (n = 4, N = 4). Data are means ± s.e.m. of independent experiments. Source data are available for this figure in the Source Data file.

Article Snippet: To overexpress TurboGFP-tagged SPNS2 for ligand binding studies, SVEC4-10 cells were transfected with 2 μg of SPNS2 ( NM_001124758 ) Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene), 2 μg of Spns2 ( NM_153060 ) Mouse Tagged ORF Clone in pCMV6-AC-GFP (# MG218672 , Origene), or as controls with 2 μg of the pCMV6-AC-GFP vector (# PS100010 , Origene) or 2 μg of glucose transporter GLUT1 (SLC2A1) ( NM_006516 ), or Human Tagged ORF Clone in pCMV6-AC-GFP (#RG222696, Origene).

Techniques: Western Blot, Plasmid Preparation, Expressing, Fluorescence, Comparison, Functional Assay, Liposomes, Control

Journal: Medicina

Article Title: Prognostic Significance of Cytoplasmic SPNS2 Expression in Patients with Oral Squamous Cell Carcinoma

doi: 10.3390/medicina57020164

Figure Lengend Snippet: Demographics and characteristics of patients with oral squamous cell carcinoma.

Article Snippet: After deparaffinization and hydration using ethanol at various concentrations, the TMAs were subjected to antigen retrieval using 0.01 M citrate buffer (pH 6.0) in a microwave, and incubated sequentially in 3% H 2 O 2 to inhibit endogenous peroxidase activity and then in 10% normal goat serum at 37 °C for 1 h. The TMAs were then mixed with solution containing polyclonal rabbit anti-human SPNS2 antibodies (Dilution 1:100×; Catalog number: NBP1-54345; Novus Biologicals, Littleton, CO, USA) at 4 °C overnight.

Techniques: Staining

Journal: Medicina

Article Title: Prognostic Significance of Cytoplasmic SPNS2 Expression in Patients with Oral Squamous Cell Carcinoma

doi: 10.3390/medicina57020164

Figure Lengend Snippet: Overexpression of SPNS2 protein in primary OSCC tissues as detected by immunohistochemical staining. Representative immunohistochemical staining of SPNS2 protein indicating negative or positive cytoplasmic expression: ( left ) Normal tissue from adjacent to the tumor, and ( right ) Primary tumor tissue. Magnification 100× ( top ) and 400× ( bottom ). Scale bars: 25 μm ( top ) and 100 μm ( bottom ).

Article Snippet: After deparaffinization and hydration using ethanol at various concentrations, the TMAs were subjected to antigen retrieval using 0.01 M citrate buffer (pH 6.0) in a microwave, and incubated sequentially in 3% H 2 O 2 to inhibit endogenous peroxidase activity and then in 10% normal goat serum at 37 °C for 1 h. The TMAs were then mixed with solution containing polyclonal rabbit anti-human SPNS2 antibodies (Dilution 1:100×; Catalog number: NBP1-54345; Novus Biologicals, Littleton, CO, USA) at 4 °C overnight.

Techniques: Over Expression, Immunohistochemical staining, Staining, Expressing

Journal: Medicina

Article Title: Prognostic Significance of Cytoplasmic SPNS2 Expression in Patients with Oral Squamous Cell Carcinoma

doi: 10.3390/medicina57020164

Figure Lengend Snippet: Clinicopathologic variables correlated with SPNS2 expression in patients with oral squamous cell carcinoma.

Article Snippet: After deparaffinization and hydration using ethanol at various concentrations, the TMAs were subjected to antigen retrieval using 0.01 M citrate buffer (pH 6.0) in a microwave, and incubated sequentially in 3% H 2 O 2 to inhibit endogenous peroxidase activity and then in 10% normal goat serum at 37 °C for 1 h. The TMAs were then mixed with solution containing polyclonal rabbit anti-human SPNS2 antibodies (Dilution 1:100×; Catalog number: NBP1-54345; Novus Biologicals, Littleton, CO, USA) at 4 °C overnight.

Techniques: Expressing, Staining

Journal: Medicina

Article Title: Prognostic Significance of Cytoplasmic SPNS2 Expression in Patients with Oral Squamous Cell Carcinoma

doi: 10.3390/medicina57020164

Figure Lengend Snippet: Kaplan–Meier survival analysis of negative and positive cytoplasmic SPNS2 protein expression in stage III/IV OSCC patients for use in log-rank tests of homogeneity using Kaplan–Meier curves. * p < 0.05.

Article Snippet: After deparaffinization and hydration using ethanol at various concentrations, the TMAs were subjected to antigen retrieval using 0.01 M citrate buffer (pH 6.0) in a microwave, and incubated sequentially in 3% H 2 O 2 to inhibit endogenous peroxidase activity and then in 10% normal goat serum at 37 °C for 1 h. The TMAs were then mixed with solution containing polyclonal rabbit anti-human SPNS2 antibodies (Dilution 1:100×; Catalog number: NBP1-54345; Novus Biologicals, Littleton, CO, USA) at 4 °C overnight.

Techniques: Expressing

Journal: Medicina

Article Title: Prognostic Significance of Cytoplasmic SPNS2 Expression in Patients with Oral Squamous Cell Carcinoma

doi: 10.3390/medicina57020164

Figure Lengend Snippet: Overall survival of III/IV stage and clinicopathologic variables of patients with oral squamous cell carcinoma using univariate and multivariate analysis.

Article Snippet: After deparaffinization and hydration using ethanol at various concentrations, the TMAs were subjected to antigen retrieval using 0.01 M citrate buffer (pH 6.0) in a microwave, and incubated sequentially in 3% H 2 O 2 to inhibit endogenous peroxidase activity and then in 10% normal goat serum at 37 °C for 1 h. The TMAs were then mixed with solution containing polyclonal rabbit anti-human SPNS2 antibodies (Dilution 1:100×; Catalog number: NBP1-54345; Novus Biologicals, Littleton, CO, USA) at 4 °C overnight.

Techniques: Expressing

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a ConSurf analysis of hSPNS2 protein. b Pairwise Alignment Scores for hSPNS2 protein and DNA sequences with putative homologs arranged according to the degree of sequence identity. c S1P levels in blood (n = 13, 15), lymph fluid (n = 10, 12), perfused liver, lung, kidney, heart (n = 3, 3). d Glucose levels in blood (n = 10,14), lymph fluid (n = 7, 13), perfused liver (n = 7, 5), lung (n = 7, 10), kidney (n = 5, 5), heart (n = 5, 5). e Glucose levels in urine (n = 7, 6), feces (n = 10, 8), tibialis anterior muscle (n = 5, 5), and gonadal adipose tissue (n = 4, 5). f Body weights (n = 20, 20). Body composition expressed as percentage of fat mass (n = 12, 12). g Food consumption, energy balance, energy expenditure, and locomotor activity (Beam breaks/h) determined with the PhenoMaster (n = 9,10; N = 3). h Blood hemoglobin concentration (n = 17, 9) and percentage of glycosylated hemoglobin, HbA1c (n = 7, 7). i Oral glucose tolerance test (GTT) and area under the curve (AUC) (n = 5, 5; N = 2). j Plasma levels of fasting insulin (n = 14, 14), glucagon (n = 8, 8) and thyroid hormone triiodothyronine (T3). Data are means ± s.e.m. k – n PET-CT imaging analysis of 2-FDG in Spns2 –/– mice. PET tracer 2-FDG (270–550 µCi) was gavaged prior to anesthetization of WT or Spns2 –/– mice for PET-CT scans. (n = 3, 3, N = 2). k , l At 60 min, tracer activity of target organs was quantified in volumes of interest (VOI). Data are percentage of whole-body activity for the right kidney (RK), heart, and bladder (Bl). l Coronal sections (0.4 mm thick) of PET images are presented according to a spectral scale for tracer activity, from red (highest), to green (intermediate), to blue (lowest). m The distribution of 2-FDG in representative WT at the specified times following gavage is shown to demonstrate the assessment of gastric emptying and intestinal absorption. St, stomach; In, intestines. n Representative images of the VOI in bladder to determine % of urinary excretion. o % of urinary excretion, gastric emptying and intestinal absorption. Data are means ± s.d. Two-tailed unpaired t-test. Source data are available for this figure in the Source Data file.

Article Snippet: Briefly, to prepare plasmid DNA templates the coding region of SPNS2 from Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene) was inserted into the expression vector pEU-E01-MCS.

Techniques: Sequencing, Activity Assay, Concentration Assay, Clinical Proteomics, Positron Emission Tomography-Computed Tomography, Imaging, Two Tailed Test

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a Immunofluorescence localization of SPNS2 (red) on plasma membrane of SVEC4-10. b Glucose increases S1P secretion by SVEC4-10 cells (n = 3, N = 3). c SVEC4-10 cells were treated with S1P lyase inhibitor A6770 (200 µM) or with SPNS2 inhibitor SLF1081851 (2 µM) and levels of S1P in cells (n = 8–10, N = 5) and medium (n = 9, N = 3) as well as glucose uptake were determined (n = 7, N = 3). d SPNS2 expression in two SPNS2 stably overexpressing SVEC4-10 cell lines generated by CRISPR activation plasmids (CTL1, SPNS2-OE1) or lentiviral activation particles (CTL2, SPNS2-OE2) and in two SPNS2 deleted SVEC4-10 cell lines (SPNS2-KO1 generated with double nickase plasmids, and SPNS2-KO2 via CRISPR/Cas9 knock-out and homology-directed repair plasmids) compared to their controls. e – h S1P levels in cells and medium, and glucose uptake were measured in SPNS2-OE1 ( e ), SPNS2-OE2 ( f ), SPNS2-KO1 ( g ), SPNS2-KO2 ( h ). (n = 6, N = 3). i , j SPNS2-OE1, SPNS2-KO1 cells and their controls were treated with S1P (500 nM), and phosphorylation of p42/44 ( i ) and glucose uptake ( j ) were determined (n = 6, N = 3). k , l Glucose uptake in SPNS2-OE1, SPNS2-KO1 and their control cells treated with insulin (100 nM) or GLUT1 inhibitor BAY-876 (10 nM) (n = 3, N = 3). Data are means ± s.d. b One-way analysis of variance test followed by Šídák’s multiple comparisons test. c – l two-tailed unpaired t-test. Source data are available for this figure in the Source Data file.

Article Snippet: Briefly, to prepare plasmid DNA templates the coding region of SPNS2 from Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene) was inserted into the expression vector pEU-E01-MCS.

Techniques: Immunofluorescence, Clinical Proteomics, Membrane, Expressing, Stable Transfection, Generated, CRISPR, Activation Assay, Knock-Out, Phospho-proteomics, Control, Two Tailed Test

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a Proliferation of Spns2 overexpressing (SPNS2-OE1) or Spns2 deleted cells (SPNS2-KO1) and controls cells. 10 5 cells were cultured in medium containing 4.5 g/L glucose without or with 500 nM S1P as indicated and cell numbers measured after 72 h (n = 3). b – f Cells were cultured in medium without or with 4.5 g/L glucose and/or 500 nM S1P as indicated. b , c Migration of cells in wound healing assays 24 h after creating a gap in a confluent monolayer and change to media containing glucose or S1P as indicated. b Representative images at 0 or 24 h after initiation of migration and ( c ) percentages of wound closures determined in cell migration assays in the presence of aphidicolin (n = 4). d – f ECIS measurements of the resistance of the indicated cells. d Representative continuous resistance measurements. e normalized endpoint resistance of the indicated cells cultured for 118 h. Resistance was normalized to the value measured at 12 h. f Normalized endpoint resistance of the indicated cells cultured for 118 h in the absence or presence of glucose and/or S1P (n = 3). g FITC-dextran leakage from the indicated cell monolayers cultured in the present of glucose (n = 3, 6). h FITC-dextran leakage from the indicated cell monolayers in the absence or presence of glucose and/or S1P (n = 4). a , c , e – h Data are means ± s.d. ns, not significant; One-way analysis of variance test followed by Sidak's multiple comparisons test or ( h ) Welch’s ANOVA multiple comparisons test. Source data are available for this figure in the Source Data file.

Article Snippet: Briefly, to prepare plasmid DNA templates the coding region of SPNS2 from Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene) was inserted into the expression vector pEU-E01-MCS.

Techniques: Cell Culture, Migration

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a Molecular dynamics simulations of the inward-facing open conformation of hSPNS2 (PDB ID 8EX4) show that S1P can slither up as its hydrocarbon tail becomes vertically aligned from its kinked structure. b Changes in the vertical distance of the S1P phosphate headgroup from its original location near S232 of SPNS2. c The upward translocation of S1P causes noticeable structural changes at the extracellular vestibule of SPNS2. d The root-mean-squared deviation (RMSD) of the SPNS2 structure as S1P slithers upward during the unbiased simulation. The set of hydrophobic residues shown in Fig. 4a pose a barrier for the upward movement illustrated by the free-energy plot shown in Supplementary Fig. . Once the phosphate group makes it past these residues, further upward movement is relatively easier, and the hydrophobic residues close in to obstruct its downward movement. e – g Snapshots of the representative, inward-facing open structures of SPNS2-S1P complexes at various times during the MD simulation in which S1P moves upward. Transient glucose-binding pockets were obtained by molecular docking of glucose into 2000 frames extracted from the 1000 ns simulation. h – j Close-up views of the glucose-binding pockets are shown in ( e – g ). Residues near the most stable glucose-binding pockets are indicated. Lower panels highlight residues within 3.5 Å of glucose in the top (site 1), central (sites 2 and 3), and bottom (site 4) glucose-binding pockets and H-bonds are indicated with dotted lines. k Glucose molecule hydrogen bonds with S1P and nearby protein residues before transitioning of glucose to the intracellular side (during 150–300 ns in the distance plot shown in m ). l Trace the glucose pathway as it enters the intracellular side from the extracellular side. m Changes in the vertical distance of glucose from its initial position at the central site 3 binding pocket as it traverses through the channel and passes into the intracellular side. Source data are available for this figure in the Source Data file.

Article Snippet: Briefly, to prepare plasmid DNA templates the coding region of SPNS2 from Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene) was inserted into the expression vector pEU-E01-MCS.

Techniques: Translocation Assay, Binding Assay

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a Representative image showing localization of SPNS2-TurboGFP and GLUT1-TurboGFP. b Temperature shift melting curves for purified hSPNS2-TurboGFP in the absence (blue) and presence of glucose (black). (n = 3, N = 3). Values are means ± s.d. c Apparent melting temperatures (T m ) for hSPNS2-TurboGFP, purified untagged hSPNS2 and purified mSPNS2-FLAG were calculated from the inflection points of the fitting curves. (n = 3, N = 3). Values are means ± s.e.m. Two-tailed unpaired t-test. d Schematic representation of the scintillation proximity assay (SPA). TurboGFP-tagged transport proteins are bound to scintillation beads. When radiolabeled glucose binds to these bead-bound transport proteins, emitted β-rays are close enough to stimulate the scintillation beads to emit light. Illustration created by Luciana Giono. e SPA signals of [ 3 H]glucose (0.8 μCi) binding to TurboGFP (vector), GLUT1-TurboGFP mSPNS2-TurboGFP, and hSPNS2-TurboGFP and its mutants E433A and T329A immobilized on the surfaces of polyvinyl toluene protein A-coated scintillation beads (500 μg per well) were measured by a scintillation counter. Values are means ± s.e.m. (n = 7; N = 2) and (n = 3; N = 3). One-way analysis of variance test followed by Dunnett’s multiple comparisons test. Source data are available for this figure in the Source Data file.

Article Snippet: Briefly, to prepare plasmid DNA templates the coding region of SPNS2 from Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene) was inserted into the expression vector pEU-E01-MCS.

Techniques: Purification, Two Tailed Test, Scintillation Proximity Assay, Binding Assay, Plasmid Preparation

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a , b Identification of key SPNS2 residues involved in glucose transport. a Western blots and representative images of localization of hSPNS2-TurboGFP and its mutants. b Glucose uptake activities of SPNS2 variants with mutations in potential key residues involved in glucose or S1P engagement. hSPN S2 , GLUT1 vector, or the indicated mutants were overexpressed in SPNS2-KO1 cells lacking endogenous SPNS2. Glucose uptake was normalized to SPNS2 expression determined by GFP fluorescence ( n = 5, N = 3). Data are means ± s.e.m. One-way analysis of variance test followed by Dunnett’s multiple comparison test. c – f Direct glucose and S1P transport by SPNS2 proteoliposomes. c Illustration of cell-free preparation of SPNS2 proteoliposomes for functional transport analysis. d Comparable levels of hSPNS2 and its variants by western blots. e , f Proteoliposomes of WT hSPNS2 and variants were loaded without or with glucose ( e ) or S1P ( f ) as indicated and uptake of 1 μM NBD-S1P ( e ) or 1 µM NBD-glucose ( f ) determined. Arbitrary units (a.u.) (n = 3, N = 3). Nonspecific uptake was measured using protein-free liposomes (empty), vector containing proteoliposomes (vector), and T1R1 containing proteoliposomes (control). (n = 3, N = 3). One-way analysis of variance test followed by Dunnett’s multiple comparisons test. g Illustration of SPNS2-mediated export of S1P out of cells while transporting glucose inward. Illustrations in panels c and g created by Luciana Giono. h – j SPNS2-mediated D-[3H]glucose uptake. h Time-dependent specific uptake of D-[ 3 H]glucose into hSPNS2-containing proteoliposomes that were loaded without or with S1P (n = 3–5, N = 3). Nonspecific uptake measured using protein-free liposomes (empty) was subtracted from the specific uptake. i Uptake of [ 3 H]glucose by hSPNS2 or empty liposomes at 40 sec (n = 4, N = 3). j Kinetics of D-glucose uptake by hSPNS2. Specific uptake measured at 40 sec was calculated by subtraction of nonspecific [ 3 H]glucose uptake by empty liposomes and fitted to a non-linear regression analysis using Michaelis–Menten enzyme kinetics plot with K M , V max , and k cat values calculated (n = 4, N = 4). Data are means ± s.e.m. of independent experiments. Source data are available for this figure in the Source Data file.

Article Snippet: Briefly, to prepare plasmid DNA templates the coding region of SPNS2 from Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene) was inserted into the expression vector pEU-E01-MCS.

Techniques: Western Blot, Plasmid Preparation, Expressing, Fluorescence, Comparison, Functional Assay, Liposomes, Control

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a ConSurf analysis of hSPNS2 protein. b Pairwise Alignment Scores for hSPNS2 protein and DNA sequences with putative homologs arranged according to the degree of sequence identity. c S1P levels in blood (n = 13, 15), lymph fluid (n = 10, 12), perfused liver, lung, kidney, heart (n = 3, 3). d Glucose levels in blood (n = 10,14), lymph fluid (n = 7, 13), perfused liver (n = 7, 5), lung (n = 7, 10), kidney (n = 5, 5), heart (n = 5, 5). e Glucose levels in urine (n = 7, 6), feces (n = 10, 8), tibialis anterior muscle (n = 5, 5), and gonadal adipose tissue (n = 4, 5). f Body weights (n = 20, 20). Body composition expressed as percentage of fat mass (n = 12, 12). g Food consumption, energy balance, energy expenditure, and locomotor activity (Beam breaks/h) determined with the PhenoMaster (n = 9,10; N = 3). h Blood hemoglobin concentration (n = 17, 9) and percentage of glycosylated hemoglobin, HbA1c (n = 7, 7). i Oral glucose tolerance test (GTT) and area under the curve (AUC) (n = 5, 5; N = 2). j Plasma levels of fasting insulin (n = 14, 14), glucagon (n = 8, 8) and thyroid hormone triiodothyronine (T3). Data are means ± s.e.m. k – n PET-CT imaging analysis of 2-FDG in Spns2 –/– mice. PET tracer 2-FDG (270–550 µCi) was gavaged prior to anesthetization of WT or Spns2 –/– mice for PET-CT scans. (n = 3, 3, N = 2). k , l At 60 min, tracer activity of target organs was quantified in volumes of interest (VOI). Data are percentage of whole-body activity for the right kidney (RK), heart, and bladder (Bl). l Coronal sections (0.4 mm thick) of PET images are presented according to a spectral scale for tracer activity, from red (highest), to green (intermediate), to blue (lowest). m The distribution of 2-FDG in representative WT at the specified times following gavage is shown to demonstrate the assessment of gastric emptying and intestinal absorption. St, stomach; In, intestines. n Representative images of the VOI in bladder to determine % of urinary excretion. o % of urinary excretion, gastric emptying and intestinal absorption. Data are means ± s.d. Two-tailed unpaired t-test. Source data are available for this figure in the Source Data file.

Article Snippet: Murine FLAG-tagged SPNS2 was purified from HCT116 cells overexpressing FLAG-tagged mouse Spns2 coding pCMV6- Entry plasmid (#MR218672, Origene).

Techniques: Sequencing, Activity Assay, Concentration Assay, Clinical Proteomics, Positron Emission Tomography-Computed Tomography, Imaging, Two Tailed Test

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a Immunofluorescence localization of SPNS2 (red) on plasma membrane of SVEC4-10. b Glucose increases S1P secretion by SVEC4-10 cells (n = 3, N = 3). c SVEC4-10 cells were treated with S1P lyase inhibitor A6770 (200 µM) or with SPNS2 inhibitor SLF1081851 (2 µM) and levels of S1P in cells (n = 8–10, N = 5) and medium (n = 9, N = 3) as well as glucose uptake were determined (n = 7, N = 3). d SPNS2 expression in two SPNS2 stably overexpressing SVEC4-10 cell lines generated by CRISPR activation plasmids (CTL1, SPNS2-OE1) or lentiviral activation particles (CTL2, SPNS2-OE2) and in two SPNS2 deleted SVEC4-10 cell lines (SPNS2-KO1 generated with double nickase plasmids, and SPNS2-KO2 via CRISPR/Cas9 knock-out and homology-directed repair plasmids) compared to their controls. e – h S1P levels in cells and medium, and glucose uptake were measured in SPNS2-OE1 ( e ), SPNS2-OE2 ( f ), SPNS2-KO1 ( g ), SPNS2-KO2 ( h ). (n = 6, N = 3). i , j SPNS2-OE1, SPNS2-KO1 cells and their controls were treated with S1P (500 nM), and phosphorylation of p42/44 ( i ) and glucose uptake ( j ) were determined (n = 6, N = 3). k , l Glucose uptake in SPNS2-OE1, SPNS2-KO1 and their control cells treated with insulin (100 nM) or GLUT1 inhibitor BAY-876 (10 nM) (n = 3, N = 3). Data are means ± s.d. b One-way analysis of variance test followed by Šídák’s multiple comparisons test. c – l two-tailed unpaired t-test. Source data are available for this figure in the Source Data file.

Article Snippet: Murine FLAG-tagged SPNS2 was purified from HCT116 cells overexpressing FLAG-tagged mouse Spns2 coding pCMV6- Entry plasmid (#MR218672, Origene).

Techniques: Immunofluorescence, Clinical Proteomics, Membrane, Expressing, Stable Transfection, Generated, CRISPR, Activation Assay, Knock-Out, Phospho-proteomics, Control, Two Tailed Test

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a Proliferation of Spns2 overexpressing (SPNS2-OE1) or Spns2 deleted cells (SPNS2-KO1) and controls cells. 10 5 cells were cultured in medium containing 4.5 g/L glucose without or with 500 nM S1P as indicated and cell numbers measured after 72 h (n = 3). b – f Cells were cultured in medium without or with 4.5 g/L glucose and/or 500 nM S1P as indicated. b , c Migration of cells in wound healing assays 24 h after creating a gap in a confluent monolayer and change to media containing glucose or S1P as indicated. b Representative images at 0 or 24 h after initiation of migration and ( c ) percentages of wound closures determined in cell migration assays in the presence of aphidicolin (n = 4). d – f ECIS measurements of the resistance of the indicated cells. d Representative continuous resistance measurements. e normalized endpoint resistance of the indicated cells cultured for 118 h. Resistance was normalized to the value measured at 12 h. f Normalized endpoint resistance of the indicated cells cultured for 118 h in the absence or presence of glucose and/or S1P (n = 3). g FITC-dextran leakage from the indicated cell monolayers cultured in the present of glucose (n = 3, 6). h FITC-dextran leakage from the indicated cell monolayers in the absence or presence of glucose and/or S1P (n = 4). a , c , e – h Data are means ± s.d. ns, not significant; One-way analysis of variance test followed by Sidak's multiple comparisons test or ( h ) Welch’s ANOVA multiple comparisons test. Source data are available for this figure in the Source Data file.

Article Snippet: Murine FLAG-tagged SPNS2 was purified from HCT116 cells overexpressing FLAG-tagged mouse Spns2 coding pCMV6- Entry plasmid (#MR218672, Origene).

Techniques: Cell Culture, Migration

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a Molecular dynamics simulations of the inward-facing open conformation of hSPNS2 (PDB ID 8EX4) show that S1P can slither up as its hydrocarbon tail becomes vertically aligned from its kinked structure. b Changes in the vertical distance of the S1P phosphate headgroup from its original location near S232 of SPNS2. c The upward translocation of S1P causes noticeable structural changes at the extracellular vestibule of SPNS2. d The root-mean-squared deviation (RMSD) of the SPNS2 structure as S1P slithers upward during the unbiased simulation. The set of hydrophobic residues shown in Fig. 4a pose a barrier for the upward movement illustrated by the free-energy plot shown in Supplementary Fig. . Once the phosphate group makes it past these residues, further upward movement is relatively easier, and the hydrophobic residues close in to obstruct its downward movement. e – g Snapshots of the representative, inward-facing open structures of SPNS2-S1P complexes at various times during the MD simulation in which S1P moves upward. Transient glucose-binding pockets were obtained by molecular docking of glucose into 2000 frames extracted from the 1000 ns simulation. h – j Close-up views of the glucose-binding pockets are shown in ( e – g ). Residues near the most stable glucose-binding pockets are indicated. Lower panels highlight residues within 3.5 Å of glucose in the top (site 1), central (sites 2 and 3), and bottom (site 4) glucose-binding pockets and H-bonds are indicated with dotted lines. k Glucose molecule hydrogen bonds with S1P and nearby protein residues before transitioning of glucose to the intracellular side (during 150–300 ns in the distance plot shown in m ). l Trace the glucose pathway as it enters the intracellular side from the extracellular side. m Changes in the vertical distance of glucose from its initial position at the central site 3 binding pocket as it traverses through the channel and passes into the intracellular side. Source data are available for this figure in the Source Data file.

Article Snippet: Murine FLAG-tagged SPNS2 was purified from HCT116 cells overexpressing FLAG-tagged mouse Spns2 coding pCMV6- Entry plasmid (#MR218672, Origene).

Techniques: Translocation Assay, Binding Assay

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a Representative image showing localization of SPNS2-TurboGFP and GLUT1-TurboGFP. b Temperature shift melting curves for purified hSPNS2-TurboGFP in the absence (blue) and presence of glucose (black). (n = 3, N = 3). Values are means ± s.d. c Apparent melting temperatures (T m ) for hSPNS2-TurboGFP, purified untagged hSPNS2 and purified mSPNS2-FLAG were calculated from the inflection points of the fitting curves. (n = 3, N = 3). Values are means ± s.e.m. Two-tailed unpaired t-test. d Schematic representation of the scintillation proximity assay (SPA). TurboGFP-tagged transport proteins are bound to scintillation beads. When radiolabeled glucose binds to these bead-bound transport proteins, emitted β-rays are close enough to stimulate the scintillation beads to emit light. Illustration created by Luciana Giono. e SPA signals of [ 3 H]glucose (0.8 μCi) binding to TurboGFP (vector), GLUT1-TurboGFP mSPNS2-TurboGFP, and hSPNS2-TurboGFP and its mutants E433A and T329A immobilized on the surfaces of polyvinyl toluene protein A-coated scintillation beads (500 μg per well) were measured by a scintillation counter. Values are means ± s.e.m. (n = 7; N = 2) and (n = 3; N = 3). One-way analysis of variance test followed by Dunnett’s multiple comparisons test. Source data are available for this figure in the Source Data file.

Article Snippet: Murine FLAG-tagged SPNS2 was purified from HCT116 cells overexpressing FLAG-tagged mouse Spns2 coding pCMV6- Entry plasmid (#MR218672, Origene).

Techniques: Purification, Two Tailed Test, Scintillation Proximity Assay, Binding Assay, Plasmid Preparation

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a , b Identification of key SPNS2 residues involved in glucose transport. a Western blots and representative images of localization of hSPNS2-TurboGFP and its mutants. b Glucose uptake activities of SPNS2 variants with mutations in potential key residues involved in glucose or S1P engagement. hSPN S2 , GLUT1 vector, or the indicated mutants were overexpressed in SPNS2-KO1 cells lacking endogenous SPNS2. Glucose uptake was normalized to SPNS2 expression determined by GFP fluorescence ( n = 5, N = 3). Data are means ± s.e.m. One-way analysis of variance test followed by Dunnett’s multiple comparison test. c – f Direct glucose and S1P transport by SPNS2 proteoliposomes. c Illustration of cell-free preparation of SPNS2 proteoliposomes for functional transport analysis. d Comparable levels of hSPNS2 and its variants by western blots. e , f Proteoliposomes of WT hSPNS2 and variants were loaded without or with glucose ( e ) or S1P ( f ) as indicated and uptake of 1 μM NBD-S1P ( e ) or 1 µM NBD-glucose ( f ) determined. Arbitrary units (a.u.) (n = 3, N = 3). Nonspecific uptake was measured using protein-free liposomes (empty), vector containing proteoliposomes (vector), and T1R1 containing proteoliposomes (control). (n = 3, N = 3). One-way analysis of variance test followed by Dunnett’s multiple comparisons test. g Illustration of SPNS2-mediated export of S1P out of cells while transporting glucose inward. Illustrations in panels c and g created by Luciana Giono. h – j SPNS2-mediated D-[3H]glucose uptake. h Time-dependent specific uptake of D-[ 3 H]glucose into hSPNS2-containing proteoliposomes that were loaded without or with S1P (n = 3–5, N = 3). Nonspecific uptake measured using protein-free liposomes (empty) was subtracted from the specific uptake. i Uptake of [ 3 H]glucose by hSPNS2 or empty liposomes at 40 sec (n = 4, N = 3). j Kinetics of D-glucose uptake by hSPNS2. Specific uptake measured at 40 sec was calculated by subtraction of nonspecific [ 3 H]glucose uptake by empty liposomes and fitted to a non-linear regression analysis using Michaelis–Menten enzyme kinetics plot with K M , V max , and k cat values calculated (n = 4, N = 4). Data are means ± s.e.m. of independent experiments. Source data are available for this figure in the Source Data file.

Article Snippet: Murine FLAG-tagged SPNS2 was purified from HCT116 cells overexpressing FLAG-tagged mouse Spns2 coding pCMV6- Entry plasmid (#MR218672, Origene).

Techniques: Western Blot, Plasmid Preparation, Expressing, Fluorescence, Comparison, Functional Assay, Liposomes, Control

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Sphingosine-1-phosphate transporter spinster homolog 2 is essential for iron-regulated metastasis of hepatocellular carcinoma.

doi: 10.1016/j.ymthe.2021.09.012

Figure Lengend Snippet: Figure 3. ID was associated with increased SPNS2 expression (A and B) N and T tissues in the liver were sectioned from the orthotopically administrated mice in Figure 2B. n = 3. (A) mRNA and (B) protein expression of SPNS2 and iron- related genes were determined. (C and D) Liver and lung tissues were sectioned from the orthotopically administrated (C) C57BL/6 mice in Figure 2B or (D) the WT (TFRC+/+) and TFRC-knockout (TFRC–/+) mice in in Figure 2E. The percentage of lymphocyte subsets and that of effective T cells in the liver (left) and lung (right) were determined by flow cytometry assay. Effective: CD 44high, CD62low. n = 3. (E) The mRNA and (F) protein expression of TFRC and SPNS2 in the human M and NM HCC tumors in Figure 1D. N = 15. Data were presented as mean ± SEM. *p < 0.05, **p < 0.01 versus IA diet, versus N mice, or versus NM. SPNS2, transporter spinster homolog 2.

Article Snippet: Construction of SPNS2 knockout C57/ BL6 mice were commissioned to Cyagen Biosciences Inc.

Techniques: Expressing, Knock-Out, Cytometry

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Sphingosine-1-phosphate transporter spinster homolog 2 is essential for iron-regulated metastasis of hepatocellular carcinoma.

doi: 10.1016/j.ymthe.2021.09.012

Figure Lengend Snippet: Figure 4. SPNS2 affected ID-enhanced HCC metastasis in an immune-independent manner in vivo (A and B) Nude mice were fed with either IA or ID diet at day 7. Human HCC Huh7 cells were orthotopically or intravenously administrated at day 0. All mice were sacrificed at day 50. Representative figures were taken at day 50. n = 3. (A) Growth of tumor cells over time in the lung of mice. (B) N and T tissues in the liver were sectioned from the orthotopically administrated mice. mRNA and protein expression of SPNS2 and iron-related genes were determined. (C and D) Growth of tumor cells over time in the lung of mice. (C) NCG mice were fed with either IA or ID diet at day 7. Human HCC Huh7 cells (upper) or mouse HCC H22 cells (lower) were orthotopically administrated at day 0. (D) NCG mice were fed with IA diet at day 7. TFRC-knockdown Huh7 cells (upper) or H22 cells (lower) and their control counterparts were orthotopically administrated at day 0. Representative figures were taken at day 50. Data were presented as mean ± SEM. *p < 0.05, **p < 0.01 versus IA diet, or versus shNC.

Article Snippet: Construction of SPNS2 knockout C57/ BL6 mice were commissioned to Cyagen Biosciences Inc.

Techniques: In Vivo, Expressing, Knockdown, Control

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Sphingosine-1-phosphate transporter spinster homolog 2 is essential for iron-regulated metastasis of hepatocellular carcinoma.

doi: 10.1016/j.ymthe.2021.09.012

Figure Lengend Snippet: Figure 5. ID enhanced HCC cell migration and invasion in vitro (A–D) Huh7 cells or primary hepatocytes were treated with either dimethyl sulfoxide (DMSO) or iron chelators, deferoxamine (DFO) and Dp44mT, for 24 h. (A) The number of dividing Huh7 cells were determined by EdU fluorescence levels. (B) Migration and invasion ability of Huh7 cells under ID. (C and D) The mRNA (left) and protein (right) expression of SPNS2 and iron-related genes in the (C) Huh7 cells and (D) primary human and mouse hepatocytes under ID. See experiment results using other HCC cell lines in Figures S8–S10. (E) Efficiency of siRNA-mediated SPNS2 knockdown in the Huh7 cells. (F) Huh7 cells were transfected with siRNA against SPSN2 and co-treated with either DMSO or DFO for 24 h. Migration and invasion abilities of SPNS2 knockdown Huh7 cells under ID. Representative figures were shown. See experiment results using other HCC cell lines in Figure S12. All in vitro experiments were performed as 3 replications. Both representative figures and quantitative data of western blot assays were shown. Data were presented as mean ± SEM. *p < 0.05, **p < 0.01 versus DMSO, or versus siNC. Edu, 5-ethynyl-20-deoxyuridine.

Article Snippet: Construction of SPNS2 knockout C57/ BL6 mice were commissioned to Cyagen Biosciences Inc.

Techniques: Migration, In Vitro, Expressing, Knockdown, Transfection, Western Blot

Journal: Molecular therapy : the journal of the American Society of Gene Therapy

Article Title: Sphingosine-1-phosphate transporter spinster homolog 2 is essential for iron-regulated metastasis of hepatocellular carcinoma.

doi: 10.1016/j.ymthe.2021.09.012

Figure Lengend Snippet: Figure 6. Inhibition of SPNS2 reduced the effect of ID on HCC pulmonary metastasis in vivo (A) Genome typing of the WT (SPNS2+/+, 409bp) and SPNS2-knockout (SPNS2/, 613 bp) mice. (B and C) WT and SPNS2-KO mice were fed with either IA or ID diet at day 7. Mouse HCC H22 cells were orthotopically administrated at day 0. All mice were sacrificed at day 50. n = 4. (B) Growth of tumor cells over time in the lung of WT and SPNS2-KO mice. Representative figures were taken at day 50. (C) The percentage of lymphocyte subsets and that of effective T cells in the liver (left) and lung (right) were determined by flow cytometry assay. Effective: CD 44high, CD62Low. (D) The SPNS2 expression in the liver of C57BL/6 mice at 1 month-post rAAV vector administration. Both representative figures and quantitative data of western blot assay were shown. n = 3. (E) Growth of orthotopically injected mouse HCC H22 cells over time in the lung of C57BL/6 mice, following rAAV8 vector administration. See protocols for in vivo imaging in Figure S5A. Representative figures were taken at day 50. n = 3. Data were presented as mean ± SEM. *p < 0.05, **p < 0.01.

Article Snippet: Construction of SPNS2 knockout C57/ BL6 mice were commissioned to Cyagen Biosciences Inc.

Techniques: Inhibition, In Vivo, Knock-Out, Cytometry, Expressing, Plasmid Preparation, Western Blot, Injection, In Vivo Imaging

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a ConSurf analysis of hSPNS2 protein. b Pairwise Alignment Scores for hSPNS2 protein and DNA sequences with putative homologs arranged according to the degree of sequence identity. c S1P levels in blood (n = 13, 15), lymph fluid (n = 10, 12), perfused liver, lung, kidney, heart (n = 3, 3). d Glucose levels in blood (n = 10,14), lymph fluid (n = 7, 13), perfused liver (n = 7, 5), lung (n = 7, 10), kidney (n = 5, 5), heart (n = 5, 5). e Glucose levels in urine (n = 7, 6), feces (n = 10, 8), tibialis anterior muscle (n = 5, 5), and gonadal adipose tissue (n = 4, 5). f Body weights (n = 20, 20). Body composition expressed as percentage of fat mass (n = 12, 12). g Food consumption, energy balance, energy expenditure, and locomotor activity (Beam breaks/h) determined with the PhenoMaster (n = 9,10; N = 3). h Blood hemoglobin concentration (n = 17, 9) and percentage of glycosylated hemoglobin, HbA1c (n = 7, 7). i Oral glucose tolerance test (GTT) and area under the curve (AUC) (n = 5, 5; N = 2). j Plasma levels of fasting insulin (n = 14, 14), glucagon (n = 8, 8) and thyroid hormone triiodothyronine (T3). Data are means ± s.e.m. k – n PET-CT imaging analysis of 2-FDG in Spns2 –/– mice. PET tracer 2-FDG (270–550 µCi) was gavaged prior to anesthetization of WT or Spns2 –/– mice for PET-CT scans. (n = 3, 3, N = 2). k , l At 60 min, tracer activity of target organs was quantified in volumes of interest (VOI). Data are percentage of whole-body activity for the right kidney (RK), heart, and bladder (Bl). l Coronal sections (0.4 mm thick) of PET images are presented according to a spectral scale for tracer activity, from red (highest), to green (intermediate), to blue (lowest). m The distribution of 2-FDG in representative WT at the specified times following gavage is shown to demonstrate the assessment of gastric emptying and intestinal absorption. St, stomach; In, intestines. n Representative images of the VOI in bladder to determine % of urinary excretion. o % of urinary excretion, gastric emptying and intestinal absorption. Data are means ± s.d. Two-tailed unpaired t-test. Source data are available for this figure in the Source Data file.

Article Snippet: To overexpress TurboGFP-tagged SPNS2 for ligand binding studies, SVEC4-10 cells were transfected with 2 μg of SPNS2 ( NM_001124758 ) Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene), 2 μg of Spns2 ( NM_153060 ) Mouse Tagged ORF Clone in pCMV6-AC-GFP (# MG218672 , Origene), or as controls with 2 μg of the pCMV6-AC-GFP vector (# PS100010 , Origene) or 2 μg of glucose transporter GLUT1 (SLC2A1) ( NM_006516 ), or Human Tagged ORF Clone in pCMV6-AC-GFP (#RG222696, Origene).

Techniques: Sequencing, Activity Assay, Concentration Assay, Clinical Proteomics, Positron Emission Tomography-Computed Tomography, Imaging, Two Tailed Test

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a Immunofluorescence localization of SPNS2 (red) on plasma membrane of SVEC4-10. b Glucose increases S1P secretion by SVEC4-10 cells (n = 3, N = 3). c SVEC4-10 cells were treated with S1P lyase inhibitor A6770 (200 µM) or with SPNS2 inhibitor SLF1081851 (2 µM) and levels of S1P in cells (n = 8–10, N = 5) and medium (n = 9, N = 3) as well as glucose uptake were determined (n = 7, N = 3). d SPNS2 expression in two SPNS2 stably overexpressing SVEC4-10 cell lines generated by CRISPR activation plasmids (CTL1, SPNS2-OE1) or lentiviral activation particles (CTL2, SPNS2-OE2) and in two SPNS2 deleted SVEC4-10 cell lines (SPNS2-KO1 generated with double nickase plasmids, and SPNS2-KO2 via CRISPR/Cas9 knock-out and homology-directed repair plasmids) compared to their controls. e – h S1P levels in cells and medium, and glucose uptake were measured in SPNS2-OE1 ( e ), SPNS2-OE2 ( f ), SPNS2-KO1 ( g ), SPNS2-KO2 ( h ). (n = 6, N = 3). i , j SPNS2-OE1, SPNS2-KO1 cells and their controls were treated with S1P (500 nM), and phosphorylation of p42/44 ( i ) and glucose uptake ( j ) were determined (n = 6, N = 3). k , l Glucose uptake in SPNS2-OE1, SPNS2-KO1 and their control cells treated with insulin (100 nM) or GLUT1 inhibitor BAY-876 (10 nM) (n = 3, N = 3). Data are means ± s.d. b One-way analysis of variance test followed by Šídák’s multiple comparisons test. c – l two-tailed unpaired t-test. Source data are available for this figure in the Source Data file.

Article Snippet: To overexpress TurboGFP-tagged SPNS2 for ligand binding studies, SVEC4-10 cells were transfected with 2 μg of SPNS2 ( NM_001124758 ) Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene), 2 μg of Spns2 ( NM_153060 ) Mouse Tagged ORF Clone in pCMV6-AC-GFP (# MG218672 , Origene), or as controls with 2 μg of the pCMV6-AC-GFP vector (# PS100010 , Origene) or 2 μg of glucose transporter GLUT1 (SLC2A1) ( NM_006516 ), or Human Tagged ORF Clone in pCMV6-AC-GFP (#RG222696, Origene).

Techniques: Immunofluorescence, Clinical Proteomics, Membrane, Expressing, Stable Transfection, Generated, CRISPR, Activation Assay, Knock-Out, Phospho-proteomics, Control, Two Tailed Test

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a Proliferation of Spns2 overexpressing (SPNS2-OE1) or Spns2 deleted cells (SPNS2-KO1) and controls cells. 10 5 cells were cultured in medium containing 4.5 g/L glucose without or with 500 nM S1P as indicated and cell numbers measured after 72 h (n = 3). b – f Cells were cultured in medium without or with 4.5 g/L glucose and/or 500 nM S1P as indicated. b , c Migration of cells in wound healing assays 24 h after creating a gap in a confluent monolayer and change to media containing glucose or S1P as indicated. b Representative images at 0 or 24 h after initiation of migration and ( c ) percentages of wound closures determined in cell migration assays in the presence of aphidicolin (n = 4). d – f ECIS measurements of the resistance of the indicated cells. d Representative continuous resistance measurements. e normalized endpoint resistance of the indicated cells cultured for 118 h. Resistance was normalized to the value measured at 12 h. f Normalized endpoint resistance of the indicated cells cultured for 118 h in the absence or presence of glucose and/or S1P (n = 3). g FITC-dextran leakage from the indicated cell monolayers cultured in the present of glucose (n = 3, 6). h FITC-dextran leakage from the indicated cell monolayers in the absence or presence of glucose and/or S1P (n = 4). a , c , e – h Data are means ± s.d. ns, not significant; One-way analysis of variance test followed by Sidak's multiple comparisons test or ( h ) Welch’s ANOVA multiple comparisons test. Source data are available for this figure in the Source Data file.

Article Snippet: To overexpress TurboGFP-tagged SPNS2 for ligand binding studies, SVEC4-10 cells were transfected with 2 μg of SPNS2 ( NM_001124758 ) Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene), 2 μg of Spns2 ( NM_153060 ) Mouse Tagged ORF Clone in pCMV6-AC-GFP (# MG218672 , Origene), or as controls with 2 μg of the pCMV6-AC-GFP vector (# PS100010 , Origene) or 2 μg of glucose transporter GLUT1 (SLC2A1) ( NM_006516 ), or Human Tagged ORF Clone in pCMV6-AC-GFP (#RG222696, Origene).

Techniques: Cell Culture, Migration

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a Molecular dynamics simulations of the inward-facing open conformation of hSPNS2 (PDB ID 8EX4) show that S1P can slither up as its hydrocarbon tail becomes vertically aligned from its kinked structure. b Changes in the vertical distance of the S1P phosphate headgroup from its original location near S232 of SPNS2. c The upward translocation of S1P causes noticeable structural changes at the extracellular vestibule of SPNS2. d The root-mean-squared deviation (RMSD) of the SPNS2 structure as S1P slithers upward during the unbiased simulation. The set of hydrophobic residues shown in Fig. 4a pose a barrier for the upward movement illustrated by the free-energy plot shown in Supplementary Fig. . Once the phosphate group makes it past these residues, further upward movement is relatively easier, and the hydrophobic residues close in to obstruct its downward movement. e – g Snapshots of the representative, inward-facing open structures of SPNS2-S1P complexes at various times during the MD simulation in which S1P moves upward. Transient glucose-binding pockets were obtained by molecular docking of glucose into 2000 frames extracted from the 1000 ns simulation. h – j Close-up views of the glucose-binding pockets are shown in ( e – g ). Residues near the most stable glucose-binding pockets are indicated. Lower panels highlight residues within 3.5 Å of glucose in the top (site 1), central (sites 2 and 3), and bottom (site 4) glucose-binding pockets and H-bonds are indicated with dotted lines. k Glucose molecule hydrogen bonds with S1P and nearby protein residues before transitioning of glucose to the intracellular side (during 150–300 ns in the distance plot shown in m ). l Trace the glucose pathway as it enters the intracellular side from the extracellular side. m Changes in the vertical distance of glucose from its initial position at the central site 3 binding pocket as it traverses through the channel and passes into the intracellular side. Source data are available for this figure in the Source Data file.

Article Snippet: To overexpress TurboGFP-tagged SPNS2 for ligand binding studies, SVEC4-10 cells were transfected with 2 μg of SPNS2 ( NM_001124758 ) Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene), 2 μg of Spns2 ( NM_153060 ) Mouse Tagged ORF Clone in pCMV6-AC-GFP (# MG218672 , Origene), or as controls with 2 μg of the pCMV6-AC-GFP vector (# PS100010 , Origene) or 2 μg of glucose transporter GLUT1 (SLC2A1) ( NM_006516 ), or Human Tagged ORF Clone in pCMV6-AC-GFP (#RG222696, Origene).

Techniques: Translocation Assay, Binding Assay

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a Representative image showing localization of SPNS2-TurboGFP and GLUT1-TurboGFP. b Temperature shift melting curves for purified hSPNS2-TurboGFP in the absence (blue) and presence of glucose (black). (n = 3, N = 3). Values are means ± s.d. c Apparent melting temperatures (T m ) for hSPNS2-TurboGFP, purified untagged hSPNS2 and purified mSPNS2-FLAG were calculated from the inflection points of the fitting curves. (n = 3, N = 3). Values are means ± s.e.m. Two-tailed unpaired t-test. d Schematic representation of the scintillation proximity assay (SPA). TurboGFP-tagged transport proteins are bound to scintillation beads. When radiolabeled glucose binds to these bead-bound transport proteins, emitted β-rays are close enough to stimulate the scintillation beads to emit light. Illustration created by Luciana Giono. e SPA signals of [ 3 H]glucose (0.8 μCi) binding to TurboGFP (vector), GLUT1-TurboGFP mSPNS2-TurboGFP, and hSPNS2-TurboGFP and its mutants E433A and T329A immobilized on the surfaces of polyvinyl toluene protein A-coated scintillation beads (500 μg per well) were measured by a scintillation counter. Values are means ± s.e.m. (n = 7; N = 2) and (n = 3; N = 3). One-way analysis of variance test followed by Dunnett’s multiple comparisons test. Source data are available for this figure in the Source Data file.

Article Snippet: To overexpress TurboGFP-tagged SPNS2 for ligand binding studies, SVEC4-10 cells were transfected with 2 μg of SPNS2 ( NM_001124758 ) Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene), 2 μg of Spns2 ( NM_153060 ) Mouse Tagged ORF Clone in pCMV6-AC-GFP (# MG218672 , Origene), or as controls with 2 μg of the pCMV6-AC-GFP vector (# PS100010 , Origene) or 2 μg of glucose transporter GLUT1 (SLC2A1) ( NM_006516 ), or Human Tagged ORF Clone in pCMV6-AC-GFP (#RG222696, Origene).

Techniques: Purification, Two Tailed Test, Scintillation Proximity Assay, Binding Assay, Plasmid Preparation

Journal: Nature Communications

Article Title: SPNS2 exports sphingosine-1-phosphate and imports glucose

doi: 10.1038/s41467-026-71659-7

Figure Lengend Snippet: a , b Identification of key SPNS2 residues involved in glucose transport. a Western blots and representative images of localization of hSPNS2-TurboGFP and its mutants. b Glucose uptake activities of SPNS2 variants with mutations in potential key residues involved in glucose or S1P engagement. hSPN S2 , GLUT1 vector, or the indicated mutants were overexpressed in SPNS2-KO1 cells lacking endogenous SPNS2. Glucose uptake was normalized to SPNS2 expression determined by GFP fluorescence ( n = 5, N = 3). Data are means ± s.e.m. One-way analysis of variance test followed by Dunnett’s multiple comparison test. c – f Direct glucose and S1P transport by SPNS2 proteoliposomes. c Illustration of cell-free preparation of SPNS2 proteoliposomes for functional transport analysis. d Comparable levels of hSPNS2 and its variants by western blots. e , f Proteoliposomes of WT hSPNS2 and variants were loaded without or with glucose ( e ) or S1P ( f ) as indicated and uptake of 1 μM NBD-S1P ( e ) or 1 µM NBD-glucose ( f ) determined. Arbitrary units (a.u.) (n = 3, N = 3). Nonspecific uptake was measured using protein-free liposomes (empty), vector containing proteoliposomes (vector), and T1R1 containing proteoliposomes (control). (n = 3, N = 3). One-way analysis of variance test followed by Dunnett’s multiple comparisons test. g Illustration of SPNS2-mediated export of S1P out of cells while transporting glucose inward. Illustrations in panels c and g created by Luciana Giono. h – j SPNS2-mediated D-[3H]glucose uptake. h Time-dependent specific uptake of D-[ 3 H]glucose into hSPNS2-containing proteoliposomes that were loaded without or with S1P (n = 3–5, N = 3). Nonspecific uptake measured using protein-free liposomes (empty) was subtracted from the specific uptake. i Uptake of [ 3 H]glucose by hSPNS2 or empty liposomes at 40 sec (n = 4, N = 3). j Kinetics of D-glucose uptake by hSPNS2. Specific uptake measured at 40 sec was calculated by subtraction of nonspecific [ 3 H]glucose uptake by empty liposomes and fitted to a non-linear regression analysis using Michaelis–Menten enzyme kinetics plot with K M , V max , and k cat values calculated (n = 4, N = 4). Data are means ± s.e.m. of independent experiments. Source data are available for this figure in the Source Data file.

Article Snippet: To overexpress TurboGFP-tagged SPNS2 for ligand binding studies, SVEC4-10 cells were transfected with 2 μg of SPNS2 ( NM_001124758 ) Human Tagged ORF Clone in pCMV6-AC-GFP (#RG225940, Origene), 2 μg of Spns2 ( NM_153060 ) Mouse Tagged ORF Clone in pCMV6-AC-GFP (# MG218672 , Origene), or as controls with 2 μg of the pCMV6-AC-GFP vector (# PS100010 , Origene) or 2 μg of glucose transporter GLUT1 (SLC2A1) ( NM_006516 ), or Human Tagged ORF Clone in pCMV6-AC-GFP (#RG222696, Origene).

Techniques: Western Blot, Plasmid Preparation, Expressing, Fluorescence, Comparison, Functional Assay, Liposomes, Control

Journal: The Journal of Biological Chemistry

Article Title: Role of Sphingosine Kinase 1 and S1P Transporter Spns2 in HGF-mediated Lamellipodia Formation in Lung Endothelium *

doi: 10.1074/jbc.M116.758946

Figure Lengend Snippet: Spns2 and extracellular S1P are essential for HGF-induced lamellipodia formation in HLMVECs. A, HLMVECs grown to ∼90% confluence in slide chambers were treated with HGF (20 ng/ml) or PBS for 30 min and probed with anti-actin and anti-Spns2 antibodies, and lamellipodia formation was examined by immunofluorescence microscopy. Co-localization of actin (red) and Spns2 (green) in lamellipodia (merge, yellow) was visualized by immunofluorescent staining as described under “Experimental Procedures.” Insets depict enhanced co-localization of actin and Spns2 in lamellipodia after HGF challenge of HLMVECs. B, the co-localization of actin and Spns2 in lamellipodia was quantified from using ImageJ software and expressed as relative pixel intensity. At least 20 cells were analyzed for each condition. C, HLMVECs grown to ∼50% confluence on slide chambers were transfected with scrambled (sc) or Spns2 siRNA (100 nm, 48 h) prior to HGF (20 ng/ml) or PBS challenge for 30 min and probed with anti-actin and anti-cortactin antibodies, and lamellipodia formation was examined by immunofluorescence microscopy with 60× oil objective. Co-localization of actin (red) and cortactin (green) in lamellipodia (merge, yellow) was visualized by immunofluorescent staining. Shown are representative images of three independent experiments. D, lamellipodia was quantified using ImageJ software and expressed as a percentage of scRNA control cells. At least 20 cells were analyzed for each condition. The values are the means ± S.E. *, significantly different compared with scRNA control cells (p < 0.01); #, significantly different in Spns2 siRNA transfected cells stimulated with HGF as compared with scRNA cells stimulated with HGF (p < 0.05). E, HLMVECs grown on 35-mm dishes to 50% confluence were transfected with scRNA or Spns2 siRNA for 48 h as indicated in C, and cell lysates were subjected to 10% SDS-PAGE and probed with anti-Spns2 antibody. Shown is a representative blot from three independent experiments. Transfection of HLMVECs with Spns2 siRNA down-regulated ∼85% of native Spns2 protein expression compared with scsiRNA transfected cells. F, HLMVECs grown to ∼90% confluence on slide chambers were preincubated with IgG (150 μg/ml) or S1P neutralizing (150 μg/ml) antibody for 1 h prior to HGF (20 ng/ml) or PBS challenge for 30 min, cells were probed with anti-actin and anti-cortactin antibodies, and lamellipodia formation was examined by immunofluorescence microscopy with 40 × objective. Shown are representative images of three independent experiments. G, lamellipodia was quantified using ImageJ software and expressed as a percentage of IgG control cells. At least 20 cells were analyzed for each condition. The values are the means ± S.E. *, significantly different compared with IgG control cells (p < 0.05); #, significantly different compared with IgG HGF-treated cells (p < 0.01). Con, control.

Article Snippet: Scrambled siRNA, siRNA for SphK1 (catalog no. sc-156038), SphK2 (catalog no. sc-39225), and Spns2 (catalog no. sc-106749) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Immunofluorescence, Microscopy, Staining, Software, Transfection, Control, SDS Page, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Role of Sphingosine Kinase 1 and S1P Transporter Spns2 in HGF-mediated Lamellipodia Formation in Lung Endothelium *

doi: 10.1074/jbc.M116.758946

Figure Lengend Snippet: Spns2 siRNA blocks HGF-induced endothelial cell migration. A, HLMVECs grown to ∼50% confluence on 35-mm dishes were transfected with scrambled (sc) or Spns2 siRNA (100 nm, 48 h) as described under “Experimental Procedures.” After 48 h, the medium was aspirated, and cells were labeled with [32P]orthophosphate (20 μCi/ml) in DMEM phosphate-free medium for 3 h prior to stimulation with vehicle or HGF (20 ng/ml) for 30 min in the presence or absence of exogenous sphingosine (1 μm). The lipids were extracted from the media (extracellular) and cells (intracellular) and analyzed for [32P]S1P accumulation by thin layer chromatography as described under “Experimental Procedures.” The values are the means ± S.E. of three independent experiments in triplicate, and the S1P formed was normalized to 105 cells. *, significantly different from control cells not treated with HGF (p < 0.01); #, significantly different from cells transfected with scRNA and stimulated with HGF with or without exogenous sphingosine (p < 0.05). B and C, HLMVECs grown on 35-mm dishes to ∼50% confluence were transfected with sc or Spns2 siRNA as described in A for 48 h. The cells were wounded as described under “Experimental Procedures,” cell migration was viewed and imaged using a confocal microscope. Wound closure was calculated at 3-, 5-, and 7-h time points after the injury. The values are the means ± S.E. of three independent experiments in triplicate. The results show that knockdown of Spns2 with siRNA attenuated HGF-induced wound closure compared with scsiRNA-treated cells challenged with HGF. Con, control.

Article Snippet: Scrambled siRNA, siRNA for SphK1 (catalog no. sc-156038), SphK2 (catalog no. sc-39225), and Spns2 (catalog no. sc-106749) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Migration, Transfection, Labeling, Thin Layer Chromatography, Control, Microscopy, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: Role of Sphingosine Kinase 1 and S1P Transporter Spns2 in HGF-mediated Lamellipodia Formation in Lung Endothelium *

doi: 10.1074/jbc.M116.758946

Figure Lengend Snippet: HGF enhances association between SphK1, Spns2, and S1P1 in HLMVECs. A, HLMVECs grown to ∼70% confluence on 100-mm dishes were infected with FLAG-tagged SphK1 adenovirus (10 multiplicity of infection) for 48 h, followed by HGF (20 ng/ml) challenge for 30 min. The cells were lysed in lysis buffer, and lysates (1 mg protein) were subjected to immunoprecipitation with anti-FLAG antibody and Western blotted with anti-FLAG, anti-S1P1, or anti-Spns2 antibody. Shown is a representative blot from three independent experiments. B, Western blots from A were subjected to image analysis, immunostaining was quantified, and data were normalized to FLAG pixels. The values are the means ± S.E. *, significantly different from cells not exposed to HGF. The results show enhanced association between SphK1, S1P1, and Spns2 after HGF stimulation (p < 0.05) compared with control cells without HGF treatment. C, HLMVECs grown to ∼90% confluence on 100-mm dishes were exposed to HGF (20 ng/ml) for indicated time points, cell lysates (1 mg of protein) were subjected to immunoprecipitation with anti-S1P1 antibody, and immunoprecipitates were subjected to Western blotting with anti-p-SphK1 (Ser225) and anti-Spns2 antibodies. Shown is a representative blot from three independent experiments. D, Western blots from C were subjected to image analysis, bands were quantified, and data were normalized to total S1P1 immunostaining. The values are the means ± S.E. *, significantly different from cells not exposed to HGF (p < 0.01); #, significantly different compared with control cells not stimulated with HGF (p < 0.05); **, significantly different compared with control cells not exposed to HGF (p < 0.001); ##, significantly different compared with control cells not stimulated with HGF (p < 0.005). The results show enhanced association of p-SphK1 and Spns2 in S1P1 immunoprecipitates after HGF stimulation of HLMVECs. E, HLMVECs grown to ∼90% confluence on 100-mm dishes were exposed to HGF (20 ng/ml) for indicated time points, cell lysates (1 mg protein) were subjected to immunoprecipitation with anti-Spns2 antibody, and immunoprecipitates were subjected to Western blotting with anti-p-SphK1 (Ser225), anti-S1P1, anti-phosphoserine, anti-phosphothreonine, and anti-Spns2 antibodies. Shown is a representative blot from three independent experiments. F, Western blots from E were subjected to image analysis, bands were quantified, and data were normalized to total Spns2 immunostaining. The values are the means ± S.E. *, significantly different from cells not exposed to HGF (p < 0.05); #, significantly different compared with control cells not stimulated with HGF (p < 0.01); **, significantly different compared with control cells not exposed to HGF (p < 0.001). The results show enhanced association of p-SphK1 and S1P1 in Spns2 immunoprecipitates after HGF stimulation of HLMVECs. Further, immunoprecipitation of Spns2 and immunostaining with anti-phosphoserine antibody confirmed that HGF stimulated Spns2 serine phosphorylation and not threonine phosphorylation. G, HLMVECs grown on slide chambers to ∼90% confluence were stimulated with HGF (20 ng/ml) for 30 min and probed with anti-SphK1, and anti-S1P1 antibodies, and lamellipodia was examined by immunofluorescence microscopy. Co-localization of SphK1 (red) and S1P1 (green) to lamellipodia (merge, yellow) is depicted. Insets depict enhanced co-localization of SphK1 with S1P1 in lamellipodia after HGF treatment. H, the co-localization of SphK1 with S1P1 was quantified using ImageJ software and expressed as relative pixel intensity. At least 20 cells were analyzed for each condition. I, HLMVECs grown on slide chambers to ∼90% confluence were stimulated with HGF (20 ng/ml) for 30 min and probed with anti-SphK1 and anti-Spns2 antibodies, and lamellipodia was examined by immunofluorescence microscopy. Co-localization of SphK1 (red) and Spns2 (green) to lamellipodia (merge, yellow) is depicted. Insets depict enhanced co-localization of SphK1 with Spns2 in lamellipodia after HGF treatment. J, the co-localization of SphK1 with Spns2 was quantified using ImageJ software and expressed as relative pixel intensity. At least 20 cells were analyzed for each condition. Con, control; IB, immunoblot.

Article Snippet: Scrambled siRNA, siRNA for SphK1 (catalog no. sc-156038), SphK2 (catalog no. sc-39225), and Spns2 (catalog no. sc-106749) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Infection, Lysis, Immunoprecipitation, Western Blot, Immunostaining, Control, Phospho-proteomics, Immunofluorescence, Microscopy, Software

Journal: The Journal of Biological Chemistry

Article Title: Role of Sphingosine Kinase 1 and S1P Transporter Spns2 in HGF-mediated Lamellipodia Formation in Lung Endothelium *

doi: 10.1074/jbc.M116.758946

Figure Lengend Snippet: Akt, but not ERK, mediates Spns2 phosphorylation. A, HLMVECs grown to ∼90% confluence on 100-mm dishes were pretreated with Akt inhibitor VIII (1 μm, 30 min) or ERK inhibitor PD98059 (1 μm, 30 min), followed by HGF stimulation (20 ng/ml) for 30 min). Cell lysates (1 mg protein) were subjected to immunoprecipitation with anti-Spns2 antibody, and the immunoprecipitates were run on 10% SDS-PAGE and Western blotted with anti-Spns2 and anti-phosphoserine antibodies. Shown are representative blots from three independent experiments. B, Western blots from A were subjected to image analysis, and the bands were quantified. The values are the means ± S.E. *, significantly different from control cells not stimulated with HGF (p < 0.05); #, significantly different in cells pretreated with Akt inhibitor and stimulated with HGF as compared with cells stimulated with HGF in the absence of Akt inhibitor (p < 0.01). C, HLMVECs grown to ∼90% confluence in 100-mm dishes were pretreated with the ERK inhibitor PD98059 (1 μm, 30 min) followed by HGF stimulation (20 ng/ml) for 30 min). The cell lysates (1 mg of protein) were subjected to immunoprecipitation with anti-Spns2 antibody, and the immunoprecipitates were run on 10% SDS-PAGE and Western blotted with anti-Spns2 and anti-phosphoserine antibodies. Shown are representative blots from three independent experiments. D, Western blots from C were subjected to image analysis, and the bands were quantified. The values are the means ± S.E. *, significantly different from control cells not stimulated with HGF (p < 0.05). E, HLMVECs grown to ∼90% confluence in slide chambers were pretreated with Akt inhibitor VII(1 μm, 30 min) prior to HGF (20 ng/ml) or PBS treatment for 30 min and probed with anti-actin and anti-cortactin antibodies, and lamellipodia formation was examined by immunofluorescence was visualized by immunofluorescent staining as described under “Experimental Procedures.” Shown is a representative image from three independent experiments. F, lamellipodia was quantified from E using image analysis software and expressed as a percentage of control. At least 20 cells were analyzed for each condition. The values are the means ± S.E. *, significantly different in cells stimulated with HGF (p < 0.05); #, significantly different in Akt inhibitor treated cells stimulated with HGF as compared with cells without Akt inhibitor (p < 0.01). Con, control; IB, immunoblot; Inh, inhibitor.

Article Snippet: Scrambled siRNA, siRNA for SphK1 (catalog no. sc-156038), SphK2 (catalog no. sc-39225), and Spns2 (catalog no. sc-106749) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Phospho-proteomics, Immunoprecipitation, SDS Page, Western Blot, Control, Immunofluorescence, Staining, Software

Journal: The Journal of Biological Chemistry

Article Title: Role of Sphingosine Kinase 1 and S1P Transporter Spns2 in HGF-mediated Lamellipodia Formation in Lung Endothelium *

doi: 10.1074/jbc.M116.758946

Figure Lengend Snippet: Proposed model on the cross-talk between HGF/c-Met and SphK1/S1P/Spns2/S1P1 signaling axis in HGF-mediated lamellipodia formation. In this model, HGF initiates activation of its receptor c-Met, which initiates phosphorylation of ERK and Akt. Activation of ERK by HGF stimulates SphK1 phosphorylation, which in turn converts sphingosine into S1P. HGF-mediated activation of PI3K/Akt pathway phosphorylates S1P transporter, Spns2, which facilitates efflux of intracellular S1P to extracellular milieu. The secreted S1P binds to its receptor S1P1 to initiate downstream pathways mediating lamellipodia formation of lung ECs. This proposed schema is a static model and does not convey the possibility that in a dynamic model the interacting partners might function differently over time.

Article Snippet: Scrambled siRNA, siRNA for SphK1 (catalog no. sc-156038), SphK2 (catalog no. sc-39225), and Spns2 (catalog no. sc-106749) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Phospho-proteomics

Journal: Journal of Clinical Investigation

Article Title: Sphingosine-1-phosphate lyase downregulation promotes colon carcinogenesis through STAT3-activated microRNAs

doi: 10.1172/jci74188

Figure Lengend Snippet: Figure 6. SPL downregulation promotes cell transformation in a STAT3-dependent manner. (A) IB of SPL and STAT3 expression in Stat3+/+ and Stat3–/– MEFs in which SPL was silenced (SPL knockdown) compared with vector-only control (C) cell lines. (B) Intracellular and (C) extracellular S1P levels in MEF lines. (D) Spns2 mRNA expression levels in MEF lines. (E) Extracellular S1P levels in Stat3+/+ SPL knockdown MEFs with or without SPNS2 siRNA and MEF Stat3+/+ control cells. (F) Cell proliferation at day 3 of MEFs grown in 0% FBS in Stat3+/+ SPL knockdown MEFs with and without SPNS2 siRNA and MEF Stat3+/+ control cells. Represen- tative bar graph from 1 individual experiment is shown. Statistical analysis was performed with data from 3 independent experiments. (G) Il6 mRNA expression levels in MEF lines. (H) Protein expression of activated JAK-2 (JAK2-P), total JAK-2 (JAK2-T), activated STAT3 (STAT3-P), and total STAT3 (STAT3-T) in control and SPL knockdown Stat3+/+ MEFs, following treatment with 125 nM S1P. (I) Cell proliferation of MEFs grown in 0% FBS. (J) Time course of tumor growth, following s.c. injection of MEF cell lines into NOD/SCID mice. Blots shown are from different gels. (K) miR-181b-1 expression and (L) Cyld expression in tumors or injection sites of NOD/SCID mice. (M) Cell proliferation at day 3 of MEFs grown in 0% FBS with or without W123 (10 μM), FTY720 (100 nm), rolipram (10 μM), NSC 74859 (25 μM), and WP1066 (1 μM) added at day 0. *P < 0.05 compared with control Stat3+/+ MEFs. #P < 0.05 compared with control SPL knockdown Stat3+/+ MEFs.

Article Snippet: MEF cells were transfected with an siRNA against Spns2 from Origene using Lipofectamine RNAiMAX from Life Technologies according to the manufacturer’s instructions for reverse transfection protocol.

Techniques: Transformation Assay, Expressing, Knockdown, Plasmid Preparation, Control, Injection