Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 2. Characteristics of the HHR thymus and differentiation status of thymocytes in the thymus. (A) Graphs show the weights and cellularities of SDR and HHR thymi at 4–5 wk of age. Three SDRs and HHRs were used for each analysis. Data are shown as mean 6 SD. *p , 0.05. (B) Histological sections of thymi from 4-wk-old SDRs and HHRs were stained with H&E. Scale bars represent 1 mm. Representative results of two SDRs and HHRs are shown. (C) The differentiation status of thymocytes was analyzed with a flow cytometer. Representative results of three SDRs and HHRs are shown. An arrow indicates a population of DP thymocytes with decreased CD4 levels. Averaged values from three independent flow cytometric analyses for the proportions of DP, CD4-SP, CD8-SP, and DN thymocytes are shown in the lower graph. Data are shown as mean 6 SD. (D) Expression levels of Cd4 and Cd8 genes in the thymus were analyzed by real-time RT-PCR. Three SDRs and HHRs were used. The level of each gene is shown relative to the value for the SDR, which is set at 1. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

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

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 3. nTreg numbers in the HHR thymus. (A) Expression levels of Cd25 and Foxp3 genes in CD4-SP thymocytes were analyzed by real-time RT- PCR. Three SDRs and HHRs were used. The level of each gene is shown relative to the value for the SDR, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (B) Upper, The proportion of CD4+CD25+ cells in the thymus was determined by flow cytometric analysis. Representative results of three SDRs and HHRs are shown. Averaged values from three independent analyses for the proportion of CD4+CD25+ cells are shown in the graph. Data are shown as mean 6 SD. Lower, The proportion of Foxp3+ cells in the CD4+CD25+ cell fraction was determined by flow cytometric analysis. Representative results of three SDRs and HHRs are shown. Averaged values from three independent analyses for the proportion of Foxp3+ cells in the CD4+CD25+ cell fraction are shown in the graph. Data are shown as mean 6 SD. *p , 0.05. (C) Upper, Estimated values for the proportion of CD4+CD25+Foxp3+ nTregs in the thymus were calculated using the values of the proportion of CD4+CD25+ cells in the thymus and those of Foxp3+ cells in the CD4+CD25+ cell fraction obtained from flow cytometric analysis and are shown in the graph. Data are shown as mean 6 SD. *p , 0.05. Lower, Estimated values for the absolute number of CD4+CD25+Foxp3+ nTregs in the thymus were calculated using the values of the proportion of CD4+CD25+ cells in the thymus and those of total thymus cell number (Fig. 2A) and are shown in the graph. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Expressing, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 5. Loss of Ly49s3 gene expression in HHR thymic cDCs. (A) Left, Genome-wide microarray CGH analysis, performed with genomic DNA from SDR and HHR livers, shows the deletion of four Ly49 family genes—Ly49s4, Ly49i4, Ly49s3, and Ly49i3—in chromosome 4 at the q42 region (shaded area). Data shown are representative of two independent analyses. Right, Genomic PCR with DNA from SDR and HHR livers was performed to confirm the deletion of DNA in this region. As an internal standard, the Ccr4 gene, located at chromosome 8q32, was used. (B) Left, RT-PCR analysis of the expression of the Ly49s3 gene was performed with total RNA from SDR and HHR thymi. As an internal standard, the Gapdh gene was used. Middle, RT- PCR analysis of Ly49s3 gene expression in DP, CD4-SP, and CD8-SP thymocytes of the SDR thymus was performed with total RNA from the cells. Note that it was not possible to isolate pure DN thymocytes by positive and/or negative selection using CD4 and CD8a microbeads because the remaining cells after the selection of DP, CD4-SP, and CD8-SP cells are a mixture of DN thymocytes and all of the other types of cells. Right, RT-PCR analysis of Ly49s3 gene expression in cDCs of SDR and HHR thymi was performed with total RNA from the cells.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Gene Expression, Genome Wide, Microarray, Reverse Transcription Polymerase Chain Reaction, Expressing, Selection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 6. Expression of the nTreg marker and MHC class II genes in the mixed-cell culture. (A) A total of 1 3 106 CD4-SP thymocytes isolated from the SDR thymus were cultured with 2.5 3 105 cDCs from the SDR thymus, and 1 3 106 CD4-SP thymocytes from the HHR thymus were cultured with 2.5 3 105 cDCs from the HHR thymus. At 3 d later, total RNA was extracted and expression levels of nTreg marker genes Foxp3, Cd25, Ctla4, and Pd-1 and MHC class II genes Rt1-Ba and Rt1-Bb were determined by real-time RT-PCR. Three independent experiments were performed for each mixed-cell culture. The level of each gene is shown relative to the value for the mixed culture of cells from the SDR thymus, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (B) To confirm the expression levels of Foxp3 and CD25, an additional mixed-cell culture experiment was performed, and the proportion of Foxp3+ or CD25+ cells was determined by flow cytometric analysis. (C) As control experiments, 1 3 106 CD4-SP thymocytes were cultured alone for 3 d and the same real-time RT-PCR analyses as above were performed. Three independent experiments were performed for each culture. The level of each gene is shown relative to the value for SDR cells, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (D) Effects of cDCs on nTreg marker gene expression. The expression levels of nTreg marker genes in mixed-cell (A) and control cultures (C) are shown in the same graph relative to the value for the SDR control culture, which is set at 1. Statistical analysis was performed between the induction ratios of gene expression. Data are shown as mean 6 SD. *p , 0.05. (E) A total of 1 3 106 CD4+CD82CD252 thymocytes isolated from SDR and HHR thymi were cultured with 2.5 3 105 cDCs isolated from SDR and HHR thymi, respectively. At 3 d later, total RNA was extracted and expression levels of nTreg marker genes Foxp3, Cd25, Ctla4, and Pd-1 were determined by real-time RT-PCR. Three independent experiments were performed for each mixed-cell culture. The level of each gene is shown relative to the value for the mixed culture of cells from the SDR thymus, which is set at 1. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Expressing, Marker, Cell Culture, Isolation, Quantitative RT-PCR, Control, Gene Expression

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Lectin-like receptor Ly49s3 on dendritic cells contributes to the differentiation of regulatory T cells in the rat thymus.

doi: 10.4049/jimmunol.1203511

Figure Lengend Snippet: FIGURE 7. Expression of the nTreg marker and MHC class II genes in the mixed-cell culture using Ly49s3-expressing HHR thymic cDCs. (A) The FLAG- tagged Ly49s3 structure is schematically represented. R: arginine residue. (B) Left, 293T cells were transfected with recombinant vectors before being packaged into the virus, and cell lysates were subjected to Western blot analysis with the anti-FLAG M2 Ab. Right, The proteins on the membrane were stained with fast green. (C) Left, cDCs from the HHR thymus were transduced with the lentiviral vector of FLAG-tagged Ly49s3, and the expression of the fusion protein on the surface of cDCs was confirmed by fluorescence microscopic observations with the anti-FLAG M2 Ab. Right, Phase contrast appearance of the identical cells shown in the left photographs. Note dendrites on the surface of the cells. The cells were suspended in buffer and all the photos were taken. Original magnification 3800. (D) A total of 2.5 3 105 cDCs isolated from the HHR thymus were transduced with the lentiviral vector of FLAG-tagged Ly49s3 (Ly) or the mock vector (Mo) and then mixed with 1 3 106 CD4-SP thymocytes isolated from the HHR thymus. At 3 d later, total RNAwas extracted and the expression levels of nTreg marker genes Foxp3, Cd25, Ctla4, and Pd-1 and MHC class II genes Rt1-Ba and Rt1-Bb were determined by real-time RT-PCR. Three independent experiments were performed for each culture. The level of each gene is shown relative to the value for the mixed culture, using cDCs transduced with the mock vector, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (E) A total of 2.5 3 105 cDCs from the HHR thymus were transduced with the lentiviral vector of FLAG-tagged Ly49s3 and then mixed with 1 3 106 CD4-SP thymocytes from the HHR thymus. Next 5 mg of anti-rat MHC class I Ab or normal IgG was added to the culture. At 3 d later, total RNA was extracted, and the expression levels of nTreg marker genes and MHC class II genes were determined by real-time RT-PCR. Three independent experiments were performed for each culture. The level of each gene is shown relative to the value for the mixed culture in the presence of normal IgG, which is set at 1. Data are shown as mean 6 SD. *p , 0.05. (F) Summary of the experiments performed using the lentiviral vector of the Ly49s3 gene and anti-MHC class I Ab. The level of each gene is shown relative to the value for mock-transduced cells, which is set at 1. Data are shown as mean 6 SD. *p , 0.05.

Article Snippet: Cell surface marker proteins were stained using FITC-conjugated mouse anti-rat CD4 (Serotec), PE-conjugated mouse anti-rat CD8a (Cedarlane Laboratories), and PE-conjugated mouse anti-rat CD25 (Cedarlane Laboratories) Abs.

Techniques: Expressing, Marker, Cell Culture, Residue, Transfection, Recombinant, Virus, Western Blot, Membrane, Staining, Transduction, Plasmid Preparation, Isolation, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Down Regulation of T Cell Receptor Expression in COPD Pulmonary CD8 Cells

doi: 10.1371/journal.pone.0071629

Figure Lengend Snippet: The 51 genes within the functional annotation cluster were input into the STRING database. A network of how each gene interacts with others was produced. A sub-group of genes which interact closely, primarily through binding to each other, was highlighted (inset image). The genes of interest were CD247; LCK: Lymphocyte-specific protein tyrosine kinase; LAT: Linker of activated T cells; SYK: spleen tyrosine kinase; VAV2: VAV2 guanine nucleotide exchange factor; VAV3: VAV3 guanine nucleotide exchange factor.

Article Snippet: TaqMan Reverse transcription PCR was performed in duplicate for each sample using 1 μl of cDNA in a 25 μl reaction in step 2 of the Verso 2-step QRT-PCR mix kit (Thermo-scientific) containing 0.5 μl of premade ABI TaqMac gene expression assays for CD247 (Hs00609525_m1, Applied Biosystems, Warrington, UK), Linker of activated T cells (LAT, Hs01065378_g1, Applied Biosystems), LCK (Hs00178427_m1, Applied Biosystems), VAV2 guanine nucleotide exchange factor (VAV2, Hs00610104_m1, Applied Biosystems) and VAV3 guanine nucleotide exchange factor (VAV3, Hs00196125_m1, Applied Biosystems) and the endogenous control was 18s ribosomal RNA (18s, Hs99999901_s1, Applied Biosystems).

Techniques: Functional Assay, Produced, Binding Assay

Journal: PLoS ONE

Article Title: Down Regulation of T Cell Receptor Expression in COPD Pulmonary CD8 Cells

doi: 10.1371/journal.pone.0071629

Figure Lengend Snippet: Gene expression levels of CD247, Leukocyte-specific protein tyrosine kinase (LCK); Linker of activated T cells (LAT); VAV2 guanine nucleotide exchange factor (VAV2) and VAV3 guanine nucleotide exchange factor (VAV3) were measured by both human U133 plus 2 affymetrix gene assay and by quantitative reverse transcription polymerase chain reaction (PCR). Fold change from blood to lung is charted. Statistically significant differences between blood and pulmonary levels measured by PCR are indicated by *p<0.05, **p<0.001. Statistically significant differences between blood and pulmonary levels measured by microarray are indicated by † Q<0.01.

Article Snippet: TaqMan Reverse transcription PCR was performed in duplicate for each sample using 1 μl of cDNA in a 25 μl reaction in step 2 of the Verso 2-step QRT-PCR mix kit (Thermo-scientific) containing 0.5 μl of premade ABI TaqMac gene expression assays for CD247 (Hs00609525_m1, Applied Biosystems, Warrington, UK), Linker of activated T cells (LAT, Hs01065378_g1, Applied Biosystems), LCK (Hs00178427_m1, Applied Biosystems), VAV2 guanine nucleotide exchange factor (VAV2, Hs00610104_m1, Applied Biosystems) and VAV3 guanine nucleotide exchange factor (VAV3, Hs00196125_m1, Applied Biosystems) and the endogenous control was 18s ribosomal RNA (18s, Hs99999901_s1, Applied Biosystems).

Techniques: Gene Expression, Gene Assay, Reverse Transcription, Polymerase Chain Reaction, Microarray

Journal: PLoS ONE

Article Title: Down Regulation of T Cell Receptor Expression in COPD Pulmonary CD8 Cells

doi: 10.1371/journal.pone.0071629

Figure Lengend Snippet: Pulmonary CD8 cells were isolated from chronic obstructive pulmonary disease (COPD, grey n = 6) and smokers with normal lung function (S, black, n = 6). Transcript levels of CD247, Leukocyte-specific protein tyrosine kinase (LCK); Linker of activated T cells (LAT); VAV2 guanine nucleotide exchange factor (VAV2) and VAV3 guanine nucleotide exchange factor (VAV3) were measured by quantitative real time polymerase chain reaction. Statistically significant differences between COPD and S are represented by *p<0.05.

Article Snippet: TaqMan Reverse transcription PCR was performed in duplicate for each sample using 1 μl of cDNA in a 25 μl reaction in step 2 of the Verso 2-step QRT-PCR mix kit (Thermo-scientific) containing 0.5 μl of premade ABI TaqMac gene expression assays for CD247 (Hs00609525_m1, Applied Biosystems, Warrington, UK), Linker of activated T cells (LAT, Hs01065378_g1, Applied Biosystems), LCK (Hs00178427_m1, Applied Biosystems), VAV2 guanine nucleotide exchange factor (VAV2, Hs00610104_m1, Applied Biosystems) and VAV3 guanine nucleotide exchange factor (VAV3, Hs00196125_m1, Applied Biosystems) and the endogenous control was 18s ribosomal RNA (18s, Hs99999901_s1, Applied Biosystems).

Techniques: Isolation, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Sphingosine-1-phosphate Phosphatase 1 Regulates Keratinocyte Differentiation and Epidermal Homeostasis * ^♦

doi: 10.1074/jbc.M113.478420

Figure Lengend Snippet: Generation of Sgpp1 knock-out mouse line by Sgpp1 gene disruption. A, intracellular metabolic pathway of S1P. B, schematic representation of the Sgpp1 gene targeting strategy. The structure of the mouse Sgpp1 locus is shown at the top, and the structure of the Sgpp1 targeted allele is shown at the bottom. Arrows F1, F2, and R represent the primers used for genotyping; arrows rt1 and rt2 represent the primers used in semiquantitative RT-PCR. C and D, mRNA expression for Sgpp1 (top gels) and Gapdh (bottom gels) determined by semiquantitative RT-PCR of various tissues from Sgpp1+/+ (WT) and Sgpp1−/− (KO) 2-day-old (C) and adult (D) mice. E and F, mRNA expression for Sgpp1 (E) and Sgpp2 (F) determined by RT-qPCR of various tissues from Sgpp1+/+ and Sgpp1−/− adult mice. LIv, liver; Spl, spleen; H, heart; Col, colon; Stom, stomach; Kid, kidney; WAT, white adipose tissue; Br, brain. The relative level of Gapdh mRNA expression in each sample was set to 1. Bars represent mean values ± S.D. Student's t test, n = 4.

Article Snippet: Alternatively, mRNA expression levels were determined by real time-quantitative PCR (RT-qPCR) using predesigned Assay-on-Demand probes and primers (Applied Biosystems, Foster City, CA) on an ABI Prism 7700 Sequence Detection System (Applied Biosystems) as follows: for mouse Sgpp1 (Mm00473016_m1); Sgpp2 (Mm01158866_m1); Sgpl1 (Mm00486079_m1); Flg (Mm01716522_m1); Lor (Mm01219285_m1); Krt10 (Mm03009921_m1); Krt14 (Mm00516876_m1); Lce1c (Mm00783426_s1); Rptn (Mm00485857_m1); Tgm3 (Mm00436999_m1); Cdsn (Mm01275230_m1); Lor (Mm01962650_s1); Hrnr (Mm00808280_s1); Spink5 (Mm00511522_m1); Ppap2a (Mm00477016_m1); Ppap2b (Mm005404516_m1); Ppap2c (Mm00479290_m1), and Gapdh (Mm99999915_g1).

Techniques: Knock-Out, Disruption, Reverse Transcription Polymerase Chain Reaction, Expressing, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Sphingosine-1-phosphate Phosphatase 1 Regulates Keratinocyte Differentiation and Epidermal Homeostasis * ^♦

doi: 10.1074/jbc.M113.478420

Figure Lengend Snippet: Sgpp1−/− pups exhibit focal desquamation and abnormal epidermal histology. A and B, Sgpp1+/+ and Sgpp1−/− mice at 0.5 days (A) and 4 days after birth (B). The arrows in B indicate sites of desquamation. C–E, histology of Sgpp1-deficient mouse skin. Paraffin sections of back skin from 2-day-old Sgpp1+/+ (C) and Sgpp1−/− (D and E) mice were stained with H&E. E represents a region of back skin undergoing peeling of the stratum corneum (marked by arrows). Bar, 200 μm. SC, stratum corneum; SL, subcorneal layer. F, stratum corneum and subcorneal layer thicknesses in 2-day-old Sgpp1+/+ and Sgpp1−/− mice. Bars represent mean values ± S.D. n = 4 for each genotype. Student's t test; *, p < 0.05; **, p < 0.01. G and H, activity of β-galactosidase, a surrogate marker for Sgpp1 expression, in 2-day-old mouse skin section. Sgpp1+/− (Sgpp1-LacZ (neo)/+) (G) and Sgpp1+/+ (+/+) (H) skin sections are shown. Arrows in G mark the keratinocyte layers that express β-galactosidase as a marker of Sgpp1 expression. This staining is absent in the +/+ skin section (H). Bar, 50 μm. SC, stratum corneum; SG, stratum granulosum; SS, stratum spinosum; SB, stratum basale.

Article Snippet: Alternatively, mRNA expression levels were determined by real time-quantitative PCR (RT-qPCR) using predesigned Assay-on-Demand probes and primers (Applied Biosystems, Foster City, CA) on an ABI Prism 7700 Sequence Detection System (Applied Biosystems) as follows: for mouse Sgpp1 (Mm00473016_m1); Sgpp2 (Mm01158866_m1); Sgpl1 (Mm00486079_m1); Flg (Mm01716522_m1); Lor (Mm01219285_m1); Krt10 (Mm03009921_m1); Krt14 (Mm00516876_m1); Lce1c (Mm00783426_s1); Rptn (Mm00485857_m1); Tgm3 (Mm00436999_m1); Cdsn (Mm01275230_m1); Lor (Mm01962650_s1); Hrnr (Mm00808280_s1); Spink5 (Mm00511522_m1); Ppap2a (Mm00477016_m1); Ppap2b (Mm005404516_m1); Ppap2c (Mm00479290_m1), and Gapdh (Mm99999915_g1).

Techniques: Staining, Activity Assay, Marker, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Sphingosine-1-phosphate Phosphatase 1 Regulates Keratinocyte Differentiation and Epidermal Homeostasis * ^♦

doi: 10.1074/jbc.M113.478420

Figure Lengend Snippet: Epidermal permeability barrier formation was normal in Sgpp1−/− mice. Epidermal barrier acquisition was tested by resistance to toluidine blue staining at early embryonic (E) day E16 (A and B), late day E16 (C and D), and at 3 days after birth (E). Embryos are viewed dorsally (A and C) and ventrally (B and D). In the 3-day-old pups (E), the arrow indicates an area of desquamation that stained with toluidine blue.

Article Snippet: Alternatively, mRNA expression levels were determined by real time-quantitative PCR (RT-qPCR) using predesigned Assay-on-Demand probes and primers (Applied Biosystems, Foster City, CA) on an ABI Prism 7700 Sequence Detection System (Applied Biosystems) as follows: for mouse Sgpp1 (Mm00473016_m1); Sgpp2 (Mm01158866_m1); Sgpl1 (Mm00486079_m1); Flg (Mm01716522_m1); Lor (Mm01219285_m1); Krt10 (Mm03009921_m1); Krt14 (Mm00516876_m1); Lce1c (Mm00783426_s1); Rptn (Mm00485857_m1); Tgm3 (Mm00436999_m1); Cdsn (Mm01275230_m1); Lor (Mm01962650_s1); Hrnr (Mm00808280_s1); Spink5 (Mm00511522_m1); Ppap2a (Mm00477016_m1); Ppap2b (Mm005404516_m1); Ppap2c (Mm00479290_m1), and Gapdh (Mm99999915_g1).

Techniques: Permeability, Staining

Journal: The Journal of Biological Chemistry

Article Title: Sphingosine-1-phosphate Phosphatase 1 Regulates Keratinocyte Differentiation and Epidermal Homeostasis * ^♦

doi: 10.1074/jbc.M113.478420

Figure Lengend Snippet: Sphingolipid profile in Sgpp1−/− mouse epidermis. The sphingolipid profile was determined by HPLC-tandem MS on epidermis isolated from Sgpp1+/+ and Sgpp1−/− 2-day-old pups. Levels of sphingosine, S1P, and dihydro-S1P (A), total ceramide (B), and individual ceramide species with different fatty-acid chain lengths (C) were determined. Values are expressed as picomoles of sphingolipid per nmol of inorganic phosphorous (Pi). Bars represent mean values ± S.D. n = 5 for each genotype. Student's t test; *, p < 0.05. ns, not significant.

Article Snippet: Alternatively, mRNA expression levels were determined by real time-quantitative PCR (RT-qPCR) using predesigned Assay-on-Demand probes and primers (Applied Biosystems, Foster City, CA) on an ABI Prism 7700 Sequence Detection System (Applied Biosystems) as follows: for mouse Sgpp1 (Mm00473016_m1); Sgpp2 (Mm01158866_m1); Sgpl1 (Mm00486079_m1); Flg (Mm01716522_m1); Lor (Mm01219285_m1); Krt10 (Mm03009921_m1); Krt14 (Mm00516876_m1); Lce1c (Mm00783426_s1); Rptn (Mm00485857_m1); Tgm3 (Mm00436999_m1); Cdsn (Mm01275230_m1); Lor (Mm01962650_s1); Hrnr (Mm00808280_s1); Spink5 (Mm00511522_m1); Ppap2a (Mm00477016_m1); Ppap2b (Mm005404516_m1); Ppap2c (Mm00479290_m1), and Gapdh (Mm99999915_g1).

Techniques: Isolation

Journal: The Journal of Biological Chemistry

Article Title: Sphingosine-1-phosphate Phosphatase 1 Regulates Keratinocyte Differentiation and Epidermal Homeostasis * ^♦

doi: 10.1074/jbc.M113.478420

Figure Lengend Snippet: Expression of proliferation and keratinocyte differentiation markers are increased in Sgpp1−/− skin. Paraffin sections of 2-day-old skin from Sgpp1+/+ and Sgpp1−/− mice were stained with antibodies against Ki67 (A and B), keratin 6 (C and D), keratin 14 (E and F), keratin 10 (G and H), loricrin (I and J), filaggrin (K and L) (green staining), and DAPI (blue). The white line represents the outer edge of the stratum corneum to mark the skin limit on the top of the image. Bar, 100 μm.

Article Snippet: Alternatively, mRNA expression levels were determined by real time-quantitative PCR (RT-qPCR) using predesigned Assay-on-Demand probes and primers (Applied Biosystems, Foster City, CA) on an ABI Prism 7700 Sequence Detection System (Applied Biosystems) as follows: for mouse Sgpp1 (Mm00473016_m1); Sgpp2 (Mm01158866_m1); Sgpl1 (Mm00486079_m1); Flg (Mm01716522_m1); Lor (Mm01219285_m1); Krt10 (Mm03009921_m1); Krt14 (Mm00516876_m1); Lce1c (Mm00783426_s1); Rptn (Mm00485857_m1); Tgm3 (Mm00436999_m1); Cdsn (Mm01275230_m1); Lor (Mm01962650_s1); Hrnr (Mm00808280_s1); Spink5 (Mm00511522_m1); Ppap2a (Mm00477016_m1); Ppap2b (Mm005404516_m1); Ppap2c (Mm00479290_m1), and Gapdh (Mm99999915_g1).

Techniques: Expressing, Staining

Journal: The Journal of Biological Chemistry

Article Title: Sphingosine-1-phosphate Phosphatase 1 Regulates Keratinocyte Differentiation and Epidermal Homeostasis * ^♦

doi: 10.1074/jbc.M113.478420

Figure Lengend Snippet: Surviving Sgpp1−/− adult mice displayed abnormal skin phenotype. A, surviving adult Sgpp1−/− mouse and its Sgpp1+/+ littermate. Arrows indicate the deep constriction rings found on the tails of Sgpp1−/− mice. B and C, paraffin sections from Sgpp1+/+ (B) and Sgpp1−/− (C) adult mouse back skin stained with H&E. Bar, 200 μm. SC, stratum corneum; SL, subcorneal layer. D–K, immunohistochemistry of Sgpp1+/+ and Sgpp1−/− adult mouse skin. Paraffin sections were stained with antibodies against keratin 6 (D and E), keratin 14 (F and G), Ki67 antigen (H and I), filaggrin (J and K) (green staining), and DAPI (blue). The white line represents the edge of the stratum corneum to mark the skin limit on the top of the image. Bar, 100 μm.

Article Snippet: Alternatively, mRNA expression levels were determined by real time-quantitative PCR (RT-qPCR) using predesigned Assay-on-Demand probes and primers (Applied Biosystems, Foster City, CA) on an ABI Prism 7700 Sequence Detection System (Applied Biosystems) as follows: for mouse Sgpp1 (Mm00473016_m1); Sgpp2 (Mm01158866_m1); Sgpl1 (Mm00486079_m1); Flg (Mm01716522_m1); Lor (Mm01219285_m1); Krt10 (Mm03009921_m1); Krt14 (Mm00516876_m1); Lce1c (Mm00783426_s1); Rptn (Mm00485857_m1); Tgm3 (Mm00436999_m1); Cdsn (Mm01275230_m1); Lor (Mm01962650_s1); Hrnr (Mm00808280_s1); Spink5 (Mm00511522_m1); Ppap2a (Mm00477016_m1); Ppap2b (Mm005404516_m1); Ppap2c (Mm00479290_m1), and Gapdh (Mm99999915_g1).

Techniques: Staining, Immunohistochemistry

Journal: The Journal of Biological Chemistry

Article Title: Sphingosine-1-phosphate Phosphatase 1 Regulates Keratinocyte Differentiation and Epidermal Homeostasis * ^♦

doi: 10.1074/jbc.M113.478420

Figure Lengend Snippet: Sphingolipid profile of Sgpp1−/− keratinocytes. Primary keratinocytes from skin of 1-day-old Sgpp1+/+ and Sgpp1−/− mice were grown in low Ca2+ media for 3–4 days. The sphingolipid profile of each culture was determined by HPLC-tandem MS. Levels of sphingosine, S1P, and dihydro-S1P (A), total ceramide (B), and individual ceramide fatty acid chain species (C) were determined. Values are expressed as picomoles of sphingolipid per nmol of inorganic phosphorous (Pi). Bars represent mean values ± S.D. n = 5 for each genotype. Student's t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001. ns, not significant.

Article Snippet: Alternatively, mRNA expression levels were determined by real time-quantitative PCR (RT-qPCR) using predesigned Assay-on-Demand probes and primers (Applied Biosystems, Foster City, CA) on an ABI Prism 7700 Sequence Detection System (Applied Biosystems) as follows: for mouse Sgpp1 (Mm00473016_m1); Sgpp2 (Mm01158866_m1); Sgpl1 (Mm00486079_m1); Flg (Mm01716522_m1); Lor (Mm01219285_m1); Krt10 (Mm03009921_m1); Krt14 (Mm00516876_m1); Lce1c (Mm00783426_s1); Rptn (Mm00485857_m1); Tgm3 (Mm00436999_m1); Cdsn (Mm01275230_m1); Lor (Mm01962650_s1); Hrnr (Mm00808280_s1); Spink5 (Mm00511522_m1); Ppap2a (Mm00477016_m1); Ppap2b (Mm005404516_m1); Ppap2c (Mm00479290_m1), and Gapdh (Mm99999915_g1).

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Sphingosine-1-phosphate Phosphatase 1 Regulates Keratinocyte Differentiation and Epidermal Homeostasis * ^♦

doi: 10.1074/jbc.M113.478420

Figure Lengend Snippet: Gene expression profile of keratinocytes in vitro. A, microarray gene expression analysis was performed with RNA from five Sgpp1+/+ and five Sgpp1−/− keratinocyte cultures, isolated from individual mice, grown in low Ca2+ media for 3–4 days. The heat map shows the raw signal values of genes that are significantly different between Sgpp1+/+ and Sgpp1−/− groups, using a cutoff of p < 0.05 and a fold change of greater than 2. B, expression levels of nine genes among the top 50 determined by microarray analysis to be overexpressed in Sgpp1−/− keratinocytes were validated by RT-qPCR. Results are expressed as mean value ± S.D. n = 6 for both genotypes. Student's t test; *, p < 0.05; **, p < 0.01.

Article Snippet: Alternatively, mRNA expression levels were determined by real time-quantitative PCR (RT-qPCR) using predesigned Assay-on-Demand probes and primers (Applied Biosystems, Foster City, CA) on an ABI Prism 7700 Sequence Detection System (Applied Biosystems) as follows: for mouse Sgpp1 (Mm00473016_m1); Sgpp2 (Mm01158866_m1); Sgpl1 (Mm00486079_m1); Flg (Mm01716522_m1); Lor (Mm01219285_m1); Krt10 (Mm03009921_m1); Krt14 (Mm00516876_m1); Lce1c (Mm00783426_s1); Rptn (Mm00485857_m1); Tgm3 (Mm00436999_m1); Cdsn (Mm01275230_m1); Lor (Mm01962650_s1); Hrnr (Mm00808280_s1); Spink5 (Mm00511522_m1); Ppap2a (Mm00477016_m1); Ppap2b (Mm005404516_m1); Ppap2c (Mm00479290_m1), and Gapdh (Mm99999915_g1).

Techniques: Gene Expression, In Vitro, Microarray, Isolation, Expressing, Quantitative RT-PCR

Journal: The Journal of Biological Chemistry

Article Title: Sphingosine-1-phosphate Phosphatase 1 Regulates Keratinocyte Differentiation and Epidermal Homeostasis * ^♦

doi: 10.1074/jbc.M113.478420

Figure Lengend Snippet: Role of S1P in keratinocyte differentiation. A, mouse keratinocytes were incubated with 10−7 m S1P for 2 days, and gene expression was determined by RT-qPCR. Data are expressed as mean value ± S.D., n = 3 for each phenotype, and is representative of three independent experiments. Student's t test; *, p < 0.05; **, p < 0.01; ***, p < 0.001. B–E, S1PR1-GPF receptor-expressing HEK293 cells were incubated for 60 min with C-DMEM (B), 10−7 m S1P (C), Sgpp1+/+ conditioned medium (D), and Sgpp1−/− conditioned medium (E). Cells were then fixed and examined using a Zeiss confocal laser scanning microscope under a ×63 oil objective. Bar, 20 μm.

Article Snippet: Alternatively, mRNA expression levels were determined by real time-quantitative PCR (RT-qPCR) using predesigned Assay-on-Demand probes and primers (Applied Biosystems, Foster City, CA) on an ABI Prism 7700 Sequence Detection System (Applied Biosystems) as follows: for mouse Sgpp1 (Mm00473016_m1); Sgpp2 (Mm01158866_m1); Sgpl1 (Mm00486079_m1); Flg (Mm01716522_m1); Lor (Mm01219285_m1); Krt10 (Mm03009921_m1); Krt14 (Mm00516876_m1); Lce1c (Mm00783426_s1); Rptn (Mm00485857_m1); Tgm3 (Mm00436999_m1); Cdsn (Mm01275230_m1); Lor (Mm01962650_s1); Hrnr (Mm00808280_s1); Spink5 (Mm00511522_m1); Ppap2a (Mm00477016_m1); Ppap2b (Mm005404516_m1); Ppap2c (Mm00479290_m1), and Gapdh (Mm99999915_g1).

Techniques: Incubation, Gene Expression, Quantitative RT-PCR, Expressing, Laser-Scanning Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Sphingosine-1-phosphate Phosphatase 1 Regulates Keratinocyte Differentiation and Epidermal Homeostasis * ^♦

doi: 10.1074/jbc.M113.478420

Figure Lengend Snippet: Mechanism for the skin phenotype found in Sgpp1−/− mice. A and B, Ca2+ distribution was visualized in situ by Calcium Green-1 staining on frozen skin sections from Sgpp1+/+ (A) and Sgpp1−/− (B) pups. Arrows indicate staining of nucleated cells in the Sgpp1−/− sections. The white line represents the outer edge of the stratum corneum to mark the skin limit on the top of the image. Bar, 100 μm. B and C, proposed mechanism. The S1P phosphatase encoded by Sgpp1 is relatively highly expressed in differentiating keratinocytes. Its deletion in keratinocytes raises S1P levels, triggering Ca2+-induced keratinocyte differentiation, resulting in a compensatory increase in keratinocyte proliferation leading to increased desquamation.

Article Snippet: Alternatively, mRNA expression levels were determined by real time-quantitative PCR (RT-qPCR) using predesigned Assay-on-Demand probes and primers (Applied Biosystems, Foster City, CA) on an ABI Prism 7700 Sequence Detection System (Applied Biosystems) as follows: for mouse Sgpp1 (Mm00473016_m1); Sgpp2 (Mm01158866_m1); Sgpl1 (Mm00486079_m1); Flg (Mm01716522_m1); Lor (Mm01219285_m1); Krt10 (Mm03009921_m1); Krt14 (Mm00516876_m1); Lce1c (Mm00783426_s1); Rptn (Mm00485857_m1); Tgm3 (Mm00436999_m1); Cdsn (Mm01275230_m1); Lor (Mm01962650_s1); Hrnr (Mm00808280_s1); Spink5 (Mm00511522_m1); Ppap2a (Mm00477016_m1); Ppap2b (Mm005404516_m1); Ppap2c (Mm00479290_m1), and Gapdh (Mm99999915_g1).

Techniques: In Situ, Staining

Journal: Nature communications

Article Title: Circulating exosomes suppress the induction of regulatory T cells via let-7i in multiple sclerosis.

doi: 10.1038/s41467-017-02406-2

Figure Lengend Snippet: Fig. 2 Exosomal miRNA profile differentiates patients with MS from healthy controls a Heat map of miRNA expression profile in the exosomes from HC and patients with MS. RNA was extracted from exosomes, which were isolated from the plasma of four HC and four patients with MS. A 3D-Gene Human miRNA Oligo chip (TORAY) was used for microarray analysis. MiRNAs with assigned identification number lower than 500 and signal intensity higher than 100 were selected and clustered based on the expression patterns of the eight samples. b Volcano plot of miRNAs in the exosomes. The expression difference of each miRNA between MS-exosome and HC-exosome is plotted on the X axis in log2 scale. p value of the difference by t test is plotted on the Y axis. Blue dots represent miRNAs with signal intensity higher than 100. Green dots represent miRNAs with identification number lower than 500 and signal intensity higher than 100. Four miRNAs pointed by arrows (red dots) were identified as candidate miRNAs upregulated in patients with MS. They were selected from green dots. c PC analysis for the MS and HC samples based on the miRNA expression profiles, using the same data as in b. d,e Quantification of the candidate miRNAs by RT-qPCR. The total amount of RNAs in the exosomes from the same amount of plasma was also examined. Based on the clinical information, patients with MS were divided into those with RRMS or SPMS and into those in remission or relapse phase. An unpaired t test was used for statistical analysis. Error bars represent the mean ± s.d. s.d. standard deviation, n.s. not significant, PC principal component, A.U. arbitrary unit, RT-qPCR reverse transcription quantitative polymerase chain reaction, RRMS relapsing-remitting MS, SPMS secondary-progressive MS

Article Snippet: Using Plasma/Serum Circulating and Exosomal RNA Purification Kit (51000, Norgen Biotek Corp., ON, Canada), total RNA was extracted from the purified exosomes that were obtained from the same amount of the plasma.

Techniques: Expressing, Isolation, Clinical Proteomics, Microarray, Quantitative RT-PCR, Standard Deviation, Reverse Transcription, Real-time Polymerase Chain Reaction