Journal: iScience

Article Title: Erythroid SLC7A5/SLC3A2 amino acid carrier controls red blood cell size and maturation

doi: 10.1016/j.isci.2022.105739

Figure Lengend Snippet: Basal expression of SLC7A5/SLC3A2 in the erythroid lineage (A) The panel represents the flow cytometry analysis showing the different splenic cell populations in basal conditions based on the surface expression of CD71 and Ter119 in control mice. SLC3A2 expression is also represented by a color code: yellow-orange representing the strongest expression and dark green-blue the weakest or absence of expression. (B) Histograms showing SLC3A2 expression in the different splenic CD71 HI /Ter119 NEG/LOW , CD71 HI /Ter119 HI , CD71 NEG /Ter119 HI and CD71 NEG/LOW /Ter119 NEG cell populations in control (in blue) and ErGFPcre Slc7a5 LoxP / LoxP mice (in green). The IgG control (in gray) is included. A representative experiment out of 23 is shown.

Article Snippet: The membranes were then blocked and probed with antibodies against: LAT1 (SLC7A5) (ANT-105, Alomone Labs, Jerusalem, Israel); β-actin (A3854, Sigma-Aldrich).

Techniques: Expressing, Flow Cytometry, Control

Journal: iScience

Article Title: Erythroid SLC7A5/SLC3A2 amino acid carrier controls red blood cell size and maturation

doi: 10.1016/j.isci.2022.105739

Figure Lengend Snippet: SLC7A5/SLC3A2 expression in splenic erythroblasts during erythropoietic stress (A) Representative flow cytometry analysis showing the different splenic cell populations in control and PHZ-treated mice based on the surface expression of CD71 and Ter119: CD71 HI /Ter119 NEG/LOW , CD71 HI /Ter119 HI , and CD71 NEG/LOW /Ter119 NEG . (B) Relative Slc7a5 mRNA expression in the spleen of control (n = 8) and ErGFPcre Slc7a5 LoxP / LoxP (n = 3) mice in baseline conditions as well as control (n = 6) and ErGFPcre Slc7a5 LoxP / LoxP (n = 5) upon PHZ treatment. Data are shown as mean ± SEM. Statistical analysis was performed using a two-tailed Student’s t test with Welch’s correction to compare the groups as indicated in the figure (∗p < 0.05). (C) Histograms showing SLC3A2 expression in each of the populations indicated in untreated control mice (in blue) and control mice treated with PHZ (in red) are shown in left panels. In the right panels, the same histograms of the left panels are shown, but overlaying the histogram of SLC3A2 expression of ErGFPcre Slc7a5 LoxP/LoxP (in green) mice treated with PHZ. The IgG-APC control is presented in gray. For A and C a representative experiment out of 9 is shown. (D) Representative flow cytometry analysis of DRAQ5 expression versus forward scatter (FSC) of the splenic CD71 HI /Ter119 HI erythroid population of PHZ-treated control mice. Two CD71 HI /Ter119 HI populations differing in DRAQ5 signal can be distinguished, DRAQ5 HI and DRAQ5 LOW . The dashed line represents unlabeled cells. (E) Representative histograms of SLC3A2 expression in CD71 HI /Ter119 HI /DRAQ5 HI and CD71 HI /Ter119 HI /DRAQ5 LOW populations in control mice treated with PHZ (in red) and ErGFPcre Slc7a5 LoxP/LoxP (in green) mice treated with PHZ are shown. The negative signal of the IgG-PyC control is shown in gray. For D and E a representative experiment out of 7 is shown.

Article Snippet: The membranes were then blocked and probed with antibodies against: LAT1 (SLC7A5) (ANT-105, Alomone Labs, Jerusalem, Israel); β-actin (A3854, Sigma-Aldrich).

Techniques: Expressing, Flow Cytometry, Control, Two Tailed Test

Journal: iScience

Article Title: Erythroid SLC7A5/SLC3A2 amino acid carrier controls red blood cell size and maturation

doi: 10.1016/j.isci.2022.105739

Figure Lengend Snippet: SLC7A5/SLC3A2 expression in circulating CD71 HI /Ter119 HI reticulocytes under anemic conditions and in response to rhEPO (A) Representative flow cytometry analysis showing circulating CD71 HI /Ter119 HI reticulocytes in PHZ-treated and control mice, as well as in mice subjected to phlebotomy. For PHZ a representative experiment out of 20 is shown. For phlebotomy, a representative experiment out of 8 is shown. (B) Representative histograms of SLC3A2 expression in circulating CD71 HI /Ter119 HI reticulocytes of untreated control mice (in blue), mice treated with PHZ or mice subjected to phlebotomy (in red), as well as ErGFPcre Slc7a5 LoxP/LoxP mice treated with PHZ or subjected to phlebotomy (in green). The IgG-APC control is represented in gray. Quantification of the mean fluorescence intensity (MFI) of SLC3A2 on circulating CD71 HI /Ter119 HI reticulocytes from control untreated mice (n = 33), control mice treated with PHZ (n = 20), ErGFPcre Slc7a5 LoxP/+ untreated mice (n = 11), ErGFPcre Slc7a5 LoxP/+ mice treated with PHZ (n = 10), ErGFPcre Slc7a5 LoxP/LoxP untreated mice (n = 16) and ErGFPcre Slc7a5 LoxP/LoxP mice treated with PHZ (n = 14). Quantification of the mean fluorescence intensity (MFI) of SLC3A2 on circulating CD71 HI /Ter119 HI reticulocytes from control mice (n = 8), control mice subjected to phlebotomy (n = 8), ErGFPcre Slc7a5 LoxP/+ untreated mice (n = 4), ErGFPcre Slc7a5 LoxP/+ mice subjected to phlebotomy (n = 4), ErGFPcre Slc7a5 LoxP / LoxP untreated mice (n = 3) and ErGFPcre Slc7a5 LoxP/LoxP mice subjected to phlebotomy (n = 3). (C) Western blot analyses of SLC7A5 relative to β-actin protein levels in circulating erythroid cells of control and ErGFPcre Slc7a5 LoxP/LoxP PHZ-treated mice. (D) L-phenylalanine uptake in isolated CD71 HI /Ter119 HI circulating reticulocytes from PHZ-treated control or ErGFPcre Slc7a5 LoxP/LoxP mice (n = 5). (E) Representative flow cytometry analysis of CD71 expression versus GYPA in healthy and anemic subjects (left panels). Histograms of SLC3A2 expression in circulating CD71 HI /GYPA HI and CD71 NEG /GYPA HI populations of control (blue) and anemic (red) subjects (right panels). A representative experiment out of 3 anemic subjects and 3 control subjects is shown. (F) Representative histograms of SLC3A2 expression in circulating CD71 HI /Ter119 HI reticulocytes of untreated control mice (blue) or mice treated with rhEPO (200U) (red). The IgG-APC control is represented in gray. Quantification of the mean fluorescence intensity (MFI) of SLC3A2 on circulating CD71 HI /Ter119 HI reticulocytes from untreated (n = 5) or rhEPO-treated control mice (n = 5), as well as from untreated (n = 3) or rhEPO-treated ErGFPcre Slc7a5 LoxP/LoxP mice (n = 5). Data are shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test when comparing more than two groups or two-tailed Student’s t test with Welch’s correction when comparing two groups (∗∗p < 0.01; ∗∗∗p < 0.005).

Article Snippet: The membranes were then blocked and probed with antibodies against: LAT1 (SLC7A5) (ANT-105, Alomone Labs, Jerusalem, Israel); β-actin (A3854, Sigma-Aldrich).

Techniques: Expressing, Flow Cytometry, Control, Fluorescence, Western Blot, Isolation, Two Tailed Test

Journal: iScience

Article Title: Erythroid SLC7A5/SLC3A2 amino acid carrier controls red blood cell size and maturation

doi: 10.1016/j.isci.2022.105739

Figure Lengend Snippet: Mice lacking SLC7A5 in the erythroid lineage develop hypererythropoietinemia and smaller circulating RBCs (A) Serum EPO protein (pg/mL) in control mice (n = 18), ErGFPcre Slc7a5 LoxP/+ mice (n = 4), and ErGFPcre Slc7a5 LoxP/LoxP mice (n = 13) under baseline conditions; in control mice (n = 18), ErGFPcre Slc7a5 LoxP/+ mice (n = 9) and ErGFPcre Slc7a5 LoxP/LoxP mice (n = 16) after PHZ treatment; and in phlebotomized control mice (n = 6), ErGFPcre Slc7a5 LoxP/+ mice (n = 4) and ErGFPcre Slc7a5 LoxP/LoxP mice (n = 6). (B) Total number of circulating RBCs in control mice (n = 28), ErGFPcre Slc7a5 LoxP/+ mice (n = 13), and ErGFPcre Slc7a5 LoxP/LoxP mice (n = 18) under basal conditions or in control mice (n = 13), ErGFPcre Slc7a5 LoxP/+ mice (n = 9), and ErGFPcre Slc7a5 LoxP/LoxP mice (n = 13) after PHZ treatment. (C) Mean corpuscular volume (MCV, μm 3 ) of circulating RBCs in control mice (n = 28), ErGFPcre Slc7a5 LoxP/+ mice (n = 13), and ErGFPcre Slc7a5 LoxP/LoxP mice (n = 18) under basal conditions, and in control mice (n = 11), ErGFPcre Slc7a5 LoxP/+ mice (n = 7), and ErGFPcre Slc7a5 LoxP/LoxP mice (n = 12) after PHZ treatment. (D) Representative images of life imaging morphology cytometry (left) and 3D-cell rendering images (right) of RBCs of ErGFPcre Slc7a5 LoxP/LoxP and control mice. The RBC populations normally distributed their areas around a central average smaller for the ErGFPcre Slc7a5 LoxP / LoxP specimens than for control mice (central panels). Graph panels show the diameter (μm), area (μm 2 ), and elongation (%) of RBCs in control, and ErGFPcre Slc7a5 LoxP/LoxP mice. Data are shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test when comparing three groups or two-tailed Student’s t test with Welch’s correction when comparing two groups (∗p < 0.05; ∗∗∗p < 0.005).

Article Snippet: The membranes were then blocked and probed with antibodies against: LAT1 (SLC7A5) (ANT-105, Alomone Labs, Jerusalem, Israel); β-actin (A3854, Sigma-Aldrich).

Techniques: Control, Imaging, Cytometry, Two Tailed Test

Journal: iScience

Article Title: Erythroid SLC7A5/SLC3A2 amino acid carrier controls red blood cell size and maturation

doi: 10.1016/j.isci.2022.105739

Figure Lengend Snippet: Erythroid-SLC7A5 deficient mice have red blood cells with lower hemoglobin content (A) Mean corpuscular hemoglobin (MCH, pg) of circulating RBCs in control mice (n = 28), ErGFPcre Slc7a5 LoxP/+ mice (n = 13), and ErGFPcre Slc7a5 LoxP/LoxP mice (n = 18) under basal conditions. (B) Representative peripheral blood stainings with May-Grünwald-Giemsa of ErGFPcre Slc7a5 LoxP/LoxP (right) and control (left) mice. (C) Representative flow cytometry histograms comparing the phospho-rpS6 Ser235/6 signal in CD71 HI /Ter119 HI splenic erythroblasts of control and temsirolimus-treated mice (left panel). Representative flow cytometry histogram comparing the phospho-rpS6 Ser235/6 signal in CD71 HI /Ter119 HI splenic erythroblasts in control, and ErGFPcre Slc7a5 LoxP/LoxP mice (right panel). Quantification of phospho-rpS6 Ser235/6 signal from control (n = 6) and temsirolimus-treated mice (n = 5) (left panel). Quantification of phospho-rpS6 Ser235/6 signal from control (n = 9), ErGFPcre Slc7a5 LoxP/+ mice (n = 8), and ErGFPcre Slc7a5 LoxP/LoxP mice (n = 7) (right panel). The dashed line represents unlabeled permeabilized cells. Data are shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test when comparing three groups or two-tailed Student’s t test with Welch’s correction when comparing two groups (∗p < 0.05; ∗∗∗p < 0.005).

Article Snippet: The membranes were then blocked and probed with antibodies against: LAT1 (SLC7A5) (ANT-105, Alomone Labs, Jerusalem, Israel); β-actin (A3854, Sigma-Aldrich).

Techniques: Control, Flow Cytometry, Two Tailed Test

Journal: iScience

Article Title: Erythroid SLC7A5/SLC3A2 amino acid carrier controls red blood cell size and maturation

doi: 10.1016/j.isci.2022.105739

Figure Lengend Snippet: Loss of SLC7A5 in the erythroid lineage increases the transition between circulating reticulocytes and mature erythrocytes (A) Flow cytometry histogram comparing the CD71 expressed by circulating Ter119 positive cells in a control, and ErGFPcre Slc7a5 LoxP/LoxP mice after PHZ treatment. Quantification of the proportion of CD71 HI /Ter119 HI , CD71 INT /Ter119 HI , and CD71 NEG /Ter119 HI in control (n = 35), ErGFPcre Slc7a5 LoxP/+ mice (n = 19), and ErGFPcre Slc7a5 LoxP/LoxP mice (n = 28) after PHZ treatment. The range of CD71 expression considered for the quantification of CD71 HI /Ter119 HI , CD71 INT /Ter119 HI , and CD71 NEG /Ter119 HI RBCs is indicated. (B) Flow cytometry histogram comparing the TMRM signal in circulating CD71 HI /Ter119 HI reticulocytes of control, and ErGFPcre Slc7a5 LoxP/LoxP mice after PHZ treatment. Quantification of TMRM signal from control (n = 7), ErGFPcre Slc7a5 LoxP/+ mice (n = 6), and ErGFPcre Slc7a5 LoxP/LoxP mice (n = 7) after PHZ treatment. The dashed line represents unlabeled cells. (C) Maximal mitochondrial oxygen consumption rate (OCR) in circulating cells of PHZ-treated mice (n = 14) and PHZ-treated ErGFPcre Slc7a5 LoxP/LoxP mice (n = 15). Data are shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test when comparing three groups or two-tailed Student’s t test with Welch’s correction when comparing two groups (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.005).

Article Snippet: The membranes were then blocked and probed with antibodies against: LAT1 (SLC7A5) (ANT-105, Alomone Labs, Jerusalem, Israel); β-actin (A3854, Sigma-Aldrich).

Techniques: Flow Cytometry, Control, Expressing, Two Tailed Test

Journal: iScience

Article Title: Erythroid SLC7A5/SLC3A2 amino acid carrier controls red blood cell size and maturation

doi: 10.1016/j.isci.2022.105739

Figure Lengend Snippet:

Article Snippet: The membranes were then blocked and probed with antibodies against: LAT1 (SLC7A5) (ANT-105, Alomone Labs, Jerusalem, Israel); β-actin (A3854, Sigma-Aldrich).

Techniques: Control, Recombinant, Enzyme-linked Immunosorbent Assay, Knock-In, Amplification, Software

Journal: PLoS ONE

Article Title: Imaging the L-Type Amino Acid Transporter-1 (LAT1) with Zr-89 ImmunoPET

doi: 10.1371/journal.pone.0077476

Figure Lengend Snippet: An illustration of the transmembrane L-type amino acid transporter-1 (LAT1) which forms a functional heterodimer with the 4Fhc heavy chain glycoprotein and is responsible for the transport of amino acids with large neutral side chains through an exchange mechanism.

Article Snippet: Isotypes of the selected anti-human LAT1 rat mAb (Ab2) used in this study are γ2a and κ. Ab2 strongly reacted with RH7777 rat hepatoma cells (kindly donated by Dr. Chiba, Mitsubishi Tanabe Pharma, Yokohama, Japan) expressing human LAT1-GFP and HCT-116 cancer cells [ , ].

Techniques: Functional Assay

Journal: PLoS ONE

Article Title: Imaging the L-Type Amino Acid Transporter-1 (LAT1) with Zr-89 ImmunoPET

doi: 10.1371/journal.pone.0077476

Figure Lengend Snippet: ( A ) Receptor saturation using varying concentrations of the radiolabeled antibody, [ 89 Zr]DFO-Ab2. ( B ) Immunoreactivity assay plot of the (total/bound) activity versus (1/[normalized cell concentration]) of [ 89 Zr]DFO-Ab2, immunoreactive fraction determined by extrapolation to infinite antigen excess (1/y-intercept). (C) Surface bound and internalized cellular accumulation of the radioimmunoconjugate over time (up to 24 h).

Article Snippet: Isotypes of the selected anti-human LAT1 rat mAb (Ab2) used in this study are γ2a and κ. Ab2 strongly reacted with RH7777 rat hepatoma cells (kindly donated by Dr. Chiba, Mitsubishi Tanabe Pharma, Yokohama, Japan) expressing human LAT1-GFP and HCT-116 cancer cells [ , ].

Techniques: Activity Assay, Concentration Assay

Journal: PLoS ONE

Article Title: Imaging the L-Type Amino Acid Transporter-1 (LAT1) with Zr-89 ImmunoPET

doi: 10.1371/journal.pone.0077476

Figure Lengend Snippet: ( A ) Representative maximum intensity projections (MIP) of small animal PET/CT images at 3 and 7 days, where tumor is clearly visualized at 3 d. The tracer uptake was blocked with 800 µg of excess Ab2-LAT-1 (7 d block). ( B ) Maximum standard uptake values confirming increased tracer accumulation in tumor lesion over time; significant decrease in accumulation was observed in the presence of blocking dose. ( C ) Immunohistochemistry showing expression of LAT-1 in excised HCT-116 tumor used in this study.

Article Snippet: Isotypes of the selected anti-human LAT1 rat mAb (Ab2) used in this study are γ2a and κ. Ab2 strongly reacted with RH7777 rat hepatoma cells (kindly donated by Dr. Chiba, Mitsubishi Tanabe Pharma, Yokohama, Japan) expressing human LAT1-GFP and HCT-116 cancer cells [ , ].

Techniques: Positron Emission Tomography-Computed Tomography, Blocking Assay, Immunohistochemistry, Expressing

Journal: PLoS ONE

Article Title: Imaging the L-Type Amino Acid Transporter-1 (LAT1) with Zr-89 ImmunoPET

doi: 10.1371/journal.pone.0077476

Figure Lengend Snippet: Biodistribution of intravenously administered 89 Zr-Ab2-DFO in selected organs of male Athymic nu/nu mice bearing subcutaneous HCT-116 tumors.

Article Snippet: Isotypes of the selected anti-human LAT1 rat mAb (Ab2) used in this study are γ2a and κ. Ab2 strongly reacted with RH7777 rat hepatoma cells (kindly donated by Dr. Chiba, Mitsubishi Tanabe Pharma, Yokohama, Japan) expressing human LAT1-GFP and HCT-116 cancer cells [ , ].

Techniques: Blocking Assay

Journal: PLoS ONE

Article Title: Imaging the L-Type Amino Acid Transporter-1 (LAT1) with Zr-89 ImmunoPET

doi: 10.1371/journal.pone.0077476

Figure Lengend Snippet: Tumor-to-organ ratios of the immunoPET tracer, [ 89 Zr]DFO-Ab2 without (blue) and with blocking doses (green) of unlabeled Ab2 at 7 days post injection with comparison to the 18 F-labeled amino acid, [ 18 F]FET, at 30 minutes after intravenous administration.

Article Snippet: Isotypes of the selected anti-human LAT1 rat mAb (Ab2) used in this study are γ2a and κ. Ab2 strongly reacted with RH7777 rat hepatoma cells (kindly donated by Dr. Chiba, Mitsubishi Tanabe Pharma, Yokohama, Japan) expressing human LAT1-GFP and HCT-116 cancer cells [ , ].

Techniques: Blocking Assay, Injection, Comparison, Labeling