Journal: Thrombosis and Haemostasis

Article Title: Endothelial LAT1 (SLC7A5) Mediates S-Nitrosothiol Import and Modulates Respiratory Sequelae of Red Blood Cell Transfusion In Vivo

doi: 10.1055/s-0044-1782182

Figure Lengend Snippet: Sequences of DNA primers for transgenic construct and for genotyping Slc7a5 transgenic mice

Article Snippet: All LAT1 protein densitometry signals were normalized to the respective values for actin (MilliporeSigma, anti-actin, monoclonal antibody, Cat. MAB1501R) as a loading control.

Techniques: Transgenic Assay, Construct, Sequencing, Mutagenesis

Journal: Thrombosis and Haemostasis

Article Title: Endothelial LAT1 (SLC7A5) Mediates S-Nitrosothiol Import and Modulates Respiratory Sequelae of Red Blood Cell Transfusion In Vivo

doi: 10.1055/s-0044-1782182

Figure Lengend Snippet: RBC sequestration and blood oxygenation changes after transfusion are LAT1-sensitive. WT (wild-type, either Cre-, vehicle-treated; ( n = 2) or Cre-, tamoxifen (Tam)-treated ( n = 6)) or LAT1 ECKD (Cre + , Tam-treated ( n = 6) mice were transfused with fresh, PKH(red)-labeled syngeneic (C57BL/6J) mouse RBCs. Typical 12-minute recordings of blood Hb O 2 saturation (SO 2 ) in mice ( A ), mean and individual peak SO 2 changes ( B ) , and fluorescence lung images ( C, D ) are shown. Images are typical of n = 3 paired experiments. Red: PKH (transfused RBCs); green: CD31; blue: DAPI. * p < 0.05 by one-way ANOVA. n = 9 F and n = 5 M mice were used in (B).

Article Snippet: All LAT1 protein densitometry signals were normalized to the respective values for actin (MilliporeSigma, anti-actin, monoclonal antibody, Cat. MAB1501R) as a loading control.

Techniques: Labeling, Fluorescence

Journal: Thrombosis and Haemostasis

Article Title: Endothelial LAT1 (SLC7A5) Mediates S-Nitrosothiol Import and Modulates Respiratory Sequelae of Red Blood Cell Transfusion In Vivo

doi: 10.1055/s-0044-1782182

Figure Lengend Snippet: Genotyping in LAT1 fl/fl ;Cdh5-Cre mice. Mouse tail genomic DNA was genotyped for endothelial cell-specific Cdh5-Cre ( upper ), the LoxP-flanked LAT1 sites ( middle ), and the LAT1 deletion ( lower ). Results in the same mice (all littermates) genotyped pre- and post-Cre induction using tamoxifen are indicated. n = 4 LAT1 fl/fl ; Cdh5-Cre + mice and n = 6 LAT1 fl/fl ; Cdh5-Cre − mice were genotyped. In mice bearing Cdh5-Cre (but not those without), floxed LAT1 was deleted after tamoxifen administration, as determined by PCR. Labels indicate the positions of the expected PCR products, including β-actin and wild-type (wt) controls. PCR, polymerase chain reaction.

Article Snippet: All LAT1 protein densitometry signals were normalized to the respective values for actin (MilliporeSigma, anti-actin, monoclonal antibody, Cat. MAB1501R) as a loading control.

Techniques: Polymerase Chain Reaction

Journal: Thrombosis and Haemostasis

Article Title: Endothelial LAT1 (SLC7A5) Mediates S-Nitrosothiol Import and Modulates Respiratory Sequelae of Red Blood Cell Transfusion In Vivo

doi: 10.1055/s-0044-1782182

Figure Lengend Snippet: Endothelial cell (EC)-specific LAT1 knockdown as determined by qPCR and Western blots. ( A ) Tamoxifen dosing and lung harvest schedule, and lung EC isolation strategy based on positive selection for CD31 and the exclusion of CD45+ and CD41+ cells. ( B ) q-PCR results. LAT1 transcript was significantly decreased in the lung ECs of LAT fl/fl ; Cdh5-Cre+ (“LAT1 ECKD ”) versus LAT fl/fl ; Cdh5-Cre − (“WT”) lung ECs. LAT1 mRNA expression was normalized to respective beta-actin. n = 6 LAT1 fl/fl ;Cdh5-Cre- and n = 4 LAT fl/fl ; Cdh5-Cre+ mice were studied. n = 5 males (M) and n = 5 females (F). ( C ) Typical Western blot image showing LAT1 knockdown at the protein level in tamoxifen-induced LAT1 ECKD (LAT f/f ; Cdh5-Cre + ) mice (relative to Cre- littermates). ( D ) Modest but significant decrease in LAT1 protein by densitometry (normalized to beta-actin). **** p = 0.0006 by unpaired t -test; * p < 0.05 by unpaired t -test. See also (available in the online version). EC, endothelial cell; qPCR, qualitative polymerase chain reaction.

Article Snippet: All LAT1 protein densitometry signals were normalized to the respective values for actin (MilliporeSigma, anti-actin, monoclonal antibody, Cat. MAB1501R) as a loading control.

Techniques: Knockdown, Western Blot, Isolation, Selection, Expressing, Polymerase Chain Reaction

Journal: Thrombosis and Haemostasis

Article Title: Endothelial LAT1 (SLC7A5) Mediates S-Nitrosothiol Import and Modulates Respiratory Sequelae of Red Blood Cell Transfusion In Vivo

doi: 10.1055/s-0044-1782182

Figure Lengend Snippet: Depressed CSNO uptake after EC-specific LAT1 deletion. ( A ) SNO levels measured by mercury-coupled photolysis-chemiluminescence (MPC) assay in WT or LAT1 ECKD MLECs isolated as described for Fig. 3 and loaded (or not) with CSNO, 250 µM. ** p < 0.01 ( n = 8 WT and n = 10 LAT1 ECKD mice were studied; n = 13 females and n = 5 males). ( B ) Gating strategy for endothelial cells (MLECs) obtained from adult mouse lungs. After excluding debris (B, top left), doublets (B, top right), and dead cells (B, bottom left), CD45 − /CD41 − and CD31 + MLECs (B, bottom right) were further assessed (relative mean fluorescence intensity). The immunostained lung cells were loaded with the SNO probe DAF-FM diacetate. Fluorescence was monitored after addition of varying [CSNO], as a function of either time ( C ; n = 1 each shown) or EC depletion of LAT1 ( D ; n = 5 per group; n = 8 F and n = 2 M). # p < 0.057 for 2-way ANOVA; *, p < 0.05 for unpaired t -test ( t -testing was performed only at the highest [CSNO]). Horizontal bars: mean. Error bars: standard error of the mean. MLEC, mouse lung endothelial cell.

Article Snippet: All LAT1 protein densitometry signals were normalized to the respective values for actin (MilliporeSigma, anti-actin, monoclonal antibody, Cat. MAB1501R) as a loading control.

Techniques: Isolation, Fluorescence

Journal: Thrombosis and Haemostasis

Article Title: Endothelial LAT1 (SLC7A5) Mediates S-Nitrosothiol Import and Modulates Respiratory Sequelae of Red Blood Cell Transfusion In Vivo

doi: 10.1055/s-0044-1782182

Figure Lengend Snippet: LAT1 distribution in the lung and brain of LAT1 ECKD and WT mice. Immunofluorescence using the biotin-streptavidin amplification (ABC method) method was performed for LAT1 (red) and CD31 (green, endothelial cells) to determine the localization of LAT1 expression within the lung and brain using confocal microscopy (Zeiss). Images indicate that the colocalization of LAT1 and CD31 (yellow; consistent with LAT1 in ECs of both capillaries and larger vessels) did not differ consistently between WT and LAT1 ECKD mice in the lung (left), but was widely suppressed in the brain (right) of LAT1 ECKD mice. LAT1 is also visible in other cell types in the lung, including airway epithelial cells and macrophages. The results are typical of lungs and brains (see also (available in the online version) (lung, n = 3 LAT1 ECKD and n = 3 WT by ABC method), (available in the online version) (brain, n = 3 LAT1 ECKD and n = 3 WT), and (available in the online version) (lung, n = 3 LAT1 ECKD and n = 3 WT, non-avidin-biotin complex method) from several mice treated with tamoxifen daily for five days as described. Blue: DAPI. Maximum intensity projection of a Z-stack after IHC. Scale bar = 20 μm.

Article Snippet: All LAT1 protein densitometry signals were normalized to the respective values for actin (MilliporeSigma, anti-actin, monoclonal antibody, Cat. MAB1501R) as a loading control.

Techniques: Immunofluorescence, Amplification, Expressing, Confocal Microscopy, Avidin-Biotin Assay