Journal: PLOS ONE

Article Title: Protein expression of the amino acid transporter SLC7A5 in tumor tissue is prognostic in early-stage colorectal cancer

doi: 10.1371/journal.pone.0298362

Figure Lengend Snippet: SLC7A5 proteins are stained in brown; cell nuclei are counter stained purple. Original magnifications: 400×. ( A ) In benign mucosa, SLC7A5 expression is negative to very weak and localized to the cytoplasmic membrane or stromal cytoplasm. ( B-F ) In colorectal cancer, SLC7A5 is detected in tumor membrane at various levels, ranging from weakly positive tumor cells to strong expression in most cells. ( G, H ) SCL7A5 expression range distribution in 522 earl-stage cancers ( G ) and 81 late-stage cancers ( H ). Total weighted H-score of 40 is used as a cut-off (vertical red line). SLC7A5 expression in B, C are considered low and in D-F are considered high.

Article Snippet: Tissue sections were incubated with SLC7A5-specific polyclonal antibodies (HPA052673, 1:50, Atlas Antibodies, Sigma) for 30 min.

Techniques: Staining, Expressing, Membrane

Journal: PLOS ONE

Article Title: Protein expression of the amino acid transporter SLC7A5 in tumor tissue is prognostic in early-stage colorectal cancer

doi: 10.1371/journal.pone.0298362

Figure Lengend Snippet: Correlation of SLC7A5 protein expression with clinicopathological features of colorectal cancer patients.

Article Snippet: Tissue sections were incubated with SLC7A5-specific polyclonal antibodies (HPA052673, 1:50, Atlas Antibodies, Sigma) for 30 min.

Techniques: Expressing

Journal: PLOS ONE

Article Title: Protein expression of the amino acid transporter SLC7A5 in tumor tissue is prognostic in early-stage colorectal cancer

doi: 10.1371/journal.pone.0298362

Figure Lengend Snippet: High expression refers to cancer tissue with immunohistochemical staining H-score ≥40. ( A, B ) In early-stage cancers, SLC7A5 high expression is significantly correlated with longer survivals and better clinical outcome. Similar significant correlations hold in the MSS (microsatellite stability) and MSI (microsatellite instability) subtypes. ( C, D ) In late-stage cancers, SLC7A5 expression is not correlated with overall or disease-free survivals.

Article Snippet: Tissue sections were incubated with SLC7A5-specific polyclonal antibodies (HPA052673, 1:50, Atlas Antibodies, Sigma) for 30 min.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: PLOS ONE

Article Title: Protein expression of the amino acid transporter SLC7A5 in tumor tissue is prognostic in early-stage colorectal cancer

doi: 10.1371/journal.pone.0298362

Figure Lengend Snippet: Univariate and multivariate analyses of patient survival in early-stage colorectal cancer.

Article Snippet: Tissue sections were incubated with SLC7A5-specific polyclonal antibodies (HPA052673, 1:50, Atlas Antibodies, Sigma) for 30 min.

Techniques: Expressing

Journal: PLOS ONE

Article Title: Protein expression of the amino acid transporter SLC7A5 in tumor tissue is prognostic in early-stage colorectal cancer

doi: 10.1371/journal.pone.0298362

Figure Lengend Snippet: The analysis was based on the mass spectrometry protein sequencing data from the CPTAC database. The Y-axis represents SLC7A5 protein level z-scores. SLC7A5 protein expression levels are significantly higher in BRAF-mutated tumors than wild-type tumors.

Article Snippet: Tissue sections were incubated with SLC7A5-specific polyclonal antibodies (HPA052673, 1:50, Atlas Antibodies, Sigma) for 30 min.

Techniques: Mass Spectrometry, Sequencing, Expressing

Journal: PLOS ONE

Article Title: Protein expression of the amino acid transporter SLC7A5 in tumor tissue is prognostic in early-stage colorectal cancer

doi: 10.1371/journal.pone.0298362

Figure Lengend Snippet: ( A ) The analysis of the SLC7A5 protein and mRNA expressions in the 86-case CPTAC colorectal cohort revealed significant consistency between protein and gene transcription levels. Spearman’s rank correlation coefficient r = 0.5360, p<0.0001. ( B ) SLC7A5 mRNA expression showed the most significant positive correlation with SLC3A2 mRNA expression among all gene transcripts in the 592-case TCGA colorectal cancer cohort. Spearman’s rank correlation coefficient r = 0.7291, p<0.0001.

Article Snippet: Tissue sections were incubated with SLC7A5-specific polyclonal antibodies (HPA052673, 1:50, Atlas Antibodies, Sigma) for 30 min.

Techniques: Expressing

Journal: PLOS ONE

Article Title: Protein expression of the amino acid transporter SLC7A5 in tumor tissue is prognostic in early-stage colorectal cancer

doi: 10.1371/journal.pone.0298362

Figure Lengend Snippet: Only those with Spearman’s rank correlation coefficient >0.4 were included in the network. Only interactions with high confidence (0.7/1.0) are shown in the figure. Amino acid transporters are colored (red). According to Gene Ontology Biological Process annotation, the SLC7A5-network is significantly enriched in ncRNA metabolic process (aqua), RNA metabolic process (blue), and cell cycle (green). Proteins/genes colored yellow are also commonly found in lymphocytes.

Article Snippet: Tissue sections were incubated with SLC7A5-specific polyclonal antibodies (HPA052673, 1:50, Atlas Antibodies, Sigma) for 30 min.

Techniques:

Journal: American journal of human genetics

Article Title: Whole exome sequencing and homozygosity mapping identify mutation in the cell polarity protein GPSM2 as the cause of nonsyndromic hearing loss DFNB82.

doi: 10.1016/j.ajhg.2010.05.010

Figure Lengend Snippet: Figure 2. Localization of Gpsm2 in the Mouse Inner Ear (A and B) Schematic representation of the cochlea (A) and utricle (B), one of the vestibular organs. The inner and outer hair cells of the cochlea make up the sensory cells; they are embedded in non- sensory supporting cells, including the Dei- ters, pillar, and Hensen cells. The utricle alsocontainshaircellsandsupporting cells. (C–J) Immunofluorescence images of the cochlea at e16.5 (C and D) and p0 (G and H) and of the utricle at e16.5 (F) and p0 (J). Gpsm2 (green) is localized at the apical surface of the hair and supporting cells, both in the cochlea and utricle and along the greater epithelial ridge (E and I) in e16.5 and p0 inner ears. In p0 cochlea, it is specifically localized at the lateral ridge of the hair cell surface. It is also localized in the pillar cells at this age. Hair cell cyto- plasm is marked by myosin VI (red), and the nuclei are marked by DAPI (blue). Paraffin embedded sections were stained with primary antibodies goat anti-myosin VI (Santa Cruz Biotechnology) and rabbit anti-Gpsm2 (ProteinTech; for validation of the antibody, see 12) and the fluores- cence-conjugated secondary antibodies donkey anti-rabbit 488 (Molecular Probes) and donkey anti-goat cy3 (Sigma). Imaging was done with an LSM 510 confocal micro- scope (Zeiss). Scale bars represent 5 mm (C, E–G, I, and J), 2 mm (D), and 2.5 mm (H). All procedures involving animals met NIH guidelines and were approved by the Animal Care and Use Committees of Tel Aviv University and the University of Washington.

Article Snippet: We evaluated inner ear sections from embryonic day 16.5 (e16.5), postnatal day 0 (p0), p15, and p60 mice by immunohistochemistry 92 The American Journal of Human Genetics 87, 90–94, July 9, 2010 with an antibody against Gpsm212 (ProteinTech) (Figure 2; Figure S2).

Techniques: Staining, Biomarker Discovery, Imaging

Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 2. YAP1 and TAZ regulate expression of SLC38A1 and SLC7A5 in HCC cells. (A) Expression pattern of SLC family genes from microarray data. Amino acid transporters are shown in red. (B-E) The indicated cells were transfected with the indicated siRNAs. Cells were used for quanti- tative RT-PCR to measure SLC38A1 and SLC7A5. Expression level was normalized with siLuc samples. (F) Quantitative RT-PCR was done with Mst1/2 knockout samples to measure connective tissue growth factor, SLC38A1, and SLC7A5 messenger RNA expression level. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test. Abbreviations: CTGF, connective tissue growth factor; Wt, wild type.

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: Expressing, Microarray, Transfection, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Knock-Out, RNA Expression

Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 4. Amino acid transporters are important for proliferation of HCC cells. (A-D) SK-Hep1 and SNU-449 HCC cells were transiently trans- fected with the indicated siRNAs, and cell proliferation rates were measured by an MTT assay at the indicated time points. Values shown were normalized to siLuc-treated cells and represent mean 6 standard deviation. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test. (E) Recovered cell growth by exogenous SLC38A1 and SLC7A5 in YAP1/TAZ-depleted SK-Hep1 cells. Myc-tagged exogenous SLC38A1 and SLC7A5 were expressed in SK-Hep1 cells. Empty expression vector pCMV6 was used as a control. Cell proliferation rates were measured by the MTT assay at 72 hours after transfection of expression vectors. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test. (F,G) Tumor weight after treat- ment with siRNA-1,2-dioleoyl-sn-glycero-3-phosphatidylcholine in a subcutaneous xenograft model (F) or an orthotopic xenograft model (G) with SK-Hep1 HCC cells. At 6 weeks after siRNA-1,2-dioleoyl-sn-glycero-3-phosphatidylcholine injection, mice were killed and tumor weights measured (n 5 10 per group). Data are presented as mean 6 standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test.

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: MTT Assay, Standard Deviation, Expressing, Plasmid Preparation, Control, Transfection, Injection

Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 5. YAP1 and TAZ directly bind to the promoters of amino acid transporters. (A) Schematic representation of SLC38A1 promoter regions for ChIP assay (top) and ChIP assay results (bottom). (B) Schematic representation of SLC7A5 promoter regions for ChIP assay (top) and ChIP assay results (bottom). ChIP assay was done in SK-Hep1 cells with indicated antibodies. Recruitment of YAP1, TAZ, and TEAD1 proteins to the SLC38A1 or SLC7A5 promoter was analyzed using primers specific to the SLC38A1 or SLC7A5 promoter. Immunoglobulin G was used as an internal control. (C,D) ChIP samples were used for quantitative RT-PCR for quantification of binding in indicated cell lines. Data are presented as mean 6 standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test. (E,F) SK-Hep1 and SNU-449 cells were transi- ently transfected with indicated complementary DNA and reporter plasmids, and luciferase activity was measured by a luminometer (Promega). Data are presented as mean 6 standard error of the mean. *P < 0.05, **P < 0.01, ***P < 0.005 by Student t test. Abbreviations: IgG, immunoglobulin G; IP, immunoprecipitation; TBS, TEAD binding site.

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: Control, Quantitative RT-PCR, Binding Assay, Transfection, Luciferase, Activity Assay, Immunoprecipitation

Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 6. mTORC1 is regulated by SLC38A1 and SLC7A5 in HCC cells. (A) SK-Hep1 and SNU-449 HCC cells were transiently trans- fected with the indicated siRNAs for 96 hours. Phosphorylation of S6K1 was assessed by western blotting with the indicated antibodies. (B) SK-Hep1 cells were treated with esterified glutamine (Gln, 4 mM) for 30 minutes after silencing of SLC38A1 with specific siRNA. Expression and phosphorylation of S6K1 and S6 were assessed in cell lysates by western blotting with indicated antibodies. (C,D) Xeno- grafted HCC tissues treated with the indicated siRNAs were used for western blot analysis with the indi- cated antibodies.

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: Phospho-proteomics, Western Blot, Expressing

Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 7. YAP1 and TAZ activate mTORC1 through regulation of SLC38A1 and SLC7A5 (A,B). SK- Hep1 (A) and SNU-449 (B) HCC cells were transiently transfected with the indicated siRNA for 96 hours. Expression of YAP1 and TAZ and phosphorylation of S6K1 and S6 were assessed by western blot- ting with the indicated antibodies. (C) SK-Hep1 cells were transfected with SLC38A1 and SLC7A5 com- plementary DNA after silencing YAP1 and TAZ with specific siRNAs. Expression and phosphorylation of S6K1 and S6 were assessed in cell lysates by western blotting with the indicated antibodies. (D) Expres- sion of Mst1, Mst2, S6K1, and S6 and phosphorylation of S6K1 and S6 were assessed in liver tissues from wild-type and Mst1/2-/- mice (4 weeks old) by western blotting with indicated antibodies. Abbrevia- tion: Wt, wild type.

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: Transfection, Expressing, Phospho-proteomics, Western Blot

Journal: Hepatology (Baltimore, Md.)

Article Title: Yes-associated protein 1 and transcriptional coactivator with PDZ-binding motif activate the mammalian target of rapamycin complex 1 pathway by regulating amino acid transporters in hepatocellular carcinoma.

doi: 10.1002/hep.28223

Figure Lengend Snippet: Fig. 8. Significance of YAP1/TAZ-mediated regulation of mTORC1 in human HCC and mouse HCC models. (A,B) Kaplan-Meier plots of overall survival of HCC patients in cohort 1. Patients were stratified according to expression level of SLC38A1 (A) or SLC7A5 (B). P values were calculated with the log-rank test. (C) Liver tumor numbers after rapamycin treatment in Mst1/2-/- mice. At 62 days after rapamycin treatment, mice were killed and tumors counted. Data are presented as mean 6 standard error of the mean. (D) Representative images of liver tissue treated with rapamycin or vehicle. (E) Expression and phosphorylation of mTOR1 downstream targets in HCC tissues assessed by western blotting with the indicated antibodies after rapamycin or vehicle treatment in Mst1/2-/-

Article Snippet: Myc-tag solute carrier family 38 member 1 (SLC38A1) and solute carrier family 7 member 5 (SLC7A5) complementary DNA clones were purchased from OriGene (RC207604 and RC203903; TrueORF Gold clone).

Techniques: Expressing, Phospho-proteomics, Western Blot

Journal: Advanced Science

Article Title: Leucine‐Dependent SLC7A5–PGAM5 Interaction Promotes Advanced Atherosclerosis Through Hindering Mitochondrial Function of Macrophages

doi: 10.1002/advs.202518359

Figure Lengend Snippet: Silencing SLC7A5 mitigates AS by enhancing the mitochondrial function of macrophages. A) H&E (left, scale: 1 mm and 500 µm) and immunofluorescent staining of SLC7A5 and CD68 (right, scale: 50 µm) of human femoral artery atherosclerotic plaques. SP, stable plaque. USP, unstable plaque. L, lumen. B) SLC7A5 mRNA levels in human advanced carotid atherosclerotic plaques (stages IV and V) compared with paired early plaques (stages I and II) in GSE43292 dataset ( n = 32/group). C,D) Immunofluorescence staining (C) and quantification of SLC7A5 (red) and CD68 (green) in the aortic sinus of Apoe −/− mice comorbid with MC38 ( n = 7‐8/group). Scale: 200 and 50 µm (high magnification). E) Schematic of the experimental design of Slc7a5 fl/fl ; Apoe −/− mice and Slc7a5 fl/fl Lyz2 ‐cre; Apoe −/− mice fed an HFD for 16‐weeks ( n = 11‐12 mice/group). F) Quantification of total (left) and Ly6C hi (right) blood monocytes from Slc7a5 ΔLyz2 and Slc7a5 fl/fl mice ( n = 6/group) G) Quantification of CCR2 expression on blood monocytes from indicated mice ( n = 6/group). H,I) H&E (H) and immunofluorescent staining of CD68 (I) of the aortic sinus of indicated mice ( n = 11‐12 mice/group). Scale in H: 250 and 100 µm (high magnification). Scale in I: 200 and 50 µm (high magnification). J) Top ten enriched KEGG pathways identified for regulated proteins with FC≤0.83, P <0.05 in RAW264.7 cells transfected with sh‐ Slc7a5 versus negative control sh‐NC. K,L) OCR (K) and ECAR (L) of BMDMs from the Slc7a5 fl/fl mice and Slc7a5 ΔLyz2 mice following 24‐h ox‐LDL (50 ug/mL) treatment ( n = 6 or 8). M) Histogram and quantification of 2‐NBDG signal in RAW264.7 cells under indicated treatment ( n = 6). N) Immunoblot of TFAM, COX4, and MT‐CO2 in RAW264.7 cells with three Slc7a5 ‐knockdown sequences (#1, #2, #3) following 24‐h ox‐LDL (50 µgml −1 ) treatment ( n ≥6/group). O) Immunofluorescence of MitoTracker‐deep red and BODIPY 493/503 in RAW264.7 cells with indicated treatment ( n ≥100 cells/group). Scale: 20 µm. Data are presented as mean ± SEM. * P <0.05, ** P <0.01, *** P <0.001, paired Student's t ‐test (B), unpaired Student's t ‐test (D, F‐I and K‐N), and Kruskal‐Wallis test (O).

Article Snippet: Breeding strategy: Slc7a5 flox/flox × Lyz2 Cre +/− mice (obtained from the Cyagen Biosciences) → Lyz2 Cre +/− ; Slc7a5 flox/flox ; then crossed with Apoe −/− to obtain Slc7a5 flox/flox Apoe −/− mice.

Techniques: Staining, Immunofluorescence, Expressing, Transfection, Negative Control, Western Blot, Knockdown

Journal: Advanced Science

Article Title: Leucine‐Dependent SLC7A5–PGAM5 Interaction Promotes Advanced Atherosclerosis Through Hindering Mitochondrial Function of Macrophages

doi: 10.1002/advs.202518359

Figure Lengend Snippet: SLC7A5 directly binds to PGAM5 in mitochondria. A) Immunoblot analysis of SLC7A5 and mitochondrial cytosol β‐tubulin, mitochondrial outer membrane TOM20, and mitochondrial inner membrane COX4 in RAW264.7 cell subcellular fractionations. B,C) Immunofluorescent staining of MitoTracker and EGFP (SLC7A5‐EGFP) in RAW264.7 cells (B) and MitoTracker and SLC7A5 in BMDMs (C). Scale: 20 µm (co‐localization analyses are shown on the right). D) Schematic of the experimental design of label‐free proteomics sequencing of co‐IP products in SLC7A5‐FLAG‐transfected RAW264.7 cells (Anti‐FLAG versus anti‐IgG. n = 3/group). E) Co‐IP of SLC7A5‐FLAG‐transfected RAW264.7 cells with FLAG antibody followed by immunoblot analysis with SLC7A5 or PGAM5 antibodies. F) PLA signal (red) from SLC7A5‐FLAG‐ and PGAM5‐HA‐ or sh‐ Pgam5 ‐transfected RAW264.7 cells ( n = 13 visual fields per group, each containing over ten cells). Scale: 20 and 5 µm (high magnification). G) Co‐IP of HA (PGAM5‐HA) and immunoblot (IB) analysis of FLAG (SLC7A5‐FLAG) in RAW264.7 cells. H) Co‐IP of FLAG (SLC7A5‐FLAG) and immunoblot (IB) analysis of HA (PGAM5‐HA) and FLAG (SLC7A5‐FLAG) in the cytosol and mitochondrial fractions of RAW264.7 cells. I) Representative images of PLA signal (green) between HA (PGAM5‐HA) and FLAG (SLC7A5‐FLAG) and Mitotracker deep red staining in RAW264.7 cells. Scales: 20 µm and 2 µm (high magnification). J) Key hydrophobic residues (colored as magenta spheres) that stabilize the SLC7A5‐PGAM5 complex in model 2. K) Schematic of full‐length PGAM5 (PGAM5‐HA) and truncation (ΔPGAM5‐HA). L) Co‐IP of SLC7A5 and HA in vehicle‐, Pgam5 ‐HA‐, and Δ Pgam5 ‐HA transfected HEK293T.

Article Snippet: Breeding strategy: Slc7a5 flox/flox × Lyz2 Cre +/− mice (obtained from the Cyagen Biosciences) → Lyz2 Cre +/− ; Slc7a5 flox/flox ; then crossed with Apoe −/− to obtain Slc7a5 flox/flox Apoe −/− mice.

Techniques: Western Blot, Membrane, Staining, Sequencing, Co-Immunoprecipitation Assay, Transfection

Journal: Advanced Science

Article Title: Leucine‐Dependent SLC7A5–PGAM5 Interaction Promotes Advanced Atherosclerosis Through Hindering Mitochondrial Function of Macrophages

doi: 10.1002/advs.202518359

Figure Lengend Snippet: PGAM5 ameliorates SLC7A5‐induced mitochondrial dysfunction through interaction with NDUFV1. A) Immunoblot analysis of OXPHOS‐related protein MT‐CO1 and PGAM5 in Pgam5 ‐HA‐transfected RAW264.7 cells. B) OCR of RAW264.7 cells that were transfected with PGAM5‐HA or NC‐HA ( n = 7/group). C) Immunoblot of OXPHOS‐related proteins (MT‐CO2 and NDUFB8) in RAW264.7 cells transfected with three Pgam5 ‐knockdown sequences ( n ≥5/group). D) Immunofluorescence staining of BODIPY 493/503 and MitoTracker‐deep red in RAW264.7 cells with the indicated treatment ( n ≥100 cells/group). Scale: 20 µm. E) OCR in RAW264.7 cells transfected with or without sh‐ Slc7a5 or sh‐ Pgam5 ( n = 5‐6/group). F) OCR in RAW264.7 cells transfected with sh‐NC or sh‐ Pgam5 in Leu‐N and Leu‐D media and treated using ox‐LDL for 24 h ( n = 5‐6/group). G) Immunofluorescent staining of SLC7A5 (red) and PGAM5 (green) in the aortic sinus of Apoe −/− mice that received PBS or MC38. SLC7A5 + PGAM5 + areas were quantified ( n = 7‐8 mice/group). Scale: 100 µm and 20 µm (high magnification). H) PLA detection of interaction between endogenous SLC7A5 and PGAM5 in macrophages derived from THP‐1 cells, with or without leucine deficiency ( n = 18‐19 visual fields per group, each containing over 30 cells). Scale:10 µm and 5 µm (high magnification). I) Immunofluorescent staining of PGAM5 and MitoTracker in THP‐1‐derived macrophages after 24‐h ox‐LDL treatment with or without 12‐h leucine deficiency ( n ≥100 cells from 19‐20 visual fields per group). Scale: 5 µm. J) Immunoblotting of subcellular fractions from HEK293T cells transfected with Pgam5 ‐HA‐ or Δ Pgam5 ‐HA, probed for HA, β‐tubulin (cytosolic marker), and TOM20 (mitochondrial outer membrane marker). K) Pgam5 ‐HA‐ or Δ Pgam5 ‐HA‐transfected SLC7A5‐overexpression RAW264.7 cells treated with ox‐LDL for 24 h. Immunoblotting of NDUFB8, MTCO1, and MTCO2 ( n = 3‐4/group). L) Schematic of the experimental design of proteomics sequencing of co‐IP products in Pgam5 ‐HA‐transfected RAW264.7 cells (Anti‐HA versus anti‐IgG). M) Co‐IP of PGAM5‐HA‐transfected RAW264.7 cells with HA antibody followed by immunoblot analysis with HA or NDUFV1 antibodies. N) Co‐IP of PGAM5‐HA‐transfected RAW264.7 cells with or without SLC7A5 knockdown. O) Immunoblot of OXPHOS‐related proteins (SDHB and NDUFB8) in RAW264.7 cells transfected with or without OE‐ Pgam5 or si‐ Ndufv1 ( n = 5/group). P) Flow cytometry of BODIPY 493/503 in indicated RAW264.7 cells treated with ox‐LDL( n = 4/group). Data are mean ± SEM. ns, not significant, * P <0.05, ** P <0.01, *** P <0.001, unpaired Student's t ‐test (B, E‐I, O, and P), Kruskal‐Wallis test (D).

Article Snippet: Breeding strategy: Slc7a5 flox/flox × Lyz2 Cre +/− mice (obtained from the Cyagen Biosciences) → Lyz2 Cre +/− ; Slc7a5 flox/flox ; then crossed with Apoe −/− to obtain Slc7a5 flox/flox Apoe −/− mice.

Techniques: Western Blot, Transfection, Knockdown, Immunofluorescence, Staining, Derivative Assay, Marker, Membrane, Over Expression, Sequencing, Co-Immunoprecipitation Assay, Flow Cytometry

Journal: Advanced Science

Article Title: Leucine‐Dependent SLC7A5–PGAM5 Interaction Promotes Advanced Atherosclerosis Through Hindering Mitochondrial Function of Macrophages

doi: 10.1002/advs.202518359

Figure Lengend Snippet: Slc7a5 deficiency increases CD5L hi macrophages by restoring mitochondrial function. A) Immunofluorescence staining and quantification of TREM2 + CD5L + cells and BODIPY 493/503 in aortic sinus plaques from PBS‐ and MC38‐treated Apoe −/− mice ( n = 9‐10/group). Scale: 100 µm, inset: 20 µm. B) Correlation between the proportion of TREM2 + CD5L + cells and BODIPY 493/503 area in plaques from indicated mice ( n = 19). C) TREM2 + CD5L + cells in human carotid plaques and their lipid content measured using ORO and BODIPY 493/503 high (a, white dotted) and low (b, yellow dotted) areas. Each point represents a 0.2 mm 2 ‐area. Pooled quantifications from 3 human plaques. Scale: 500 and 100 µm (high magnification) D) Venn diagram illustrating the common genes of differentially‐expressed proteins (sh‐ Slc7a5 versus sh‐NC, FC≥1.2 or FC≤0.83, P <0.05) in RAW264.7 cells and marker genes of the Trem2 + Cd5l + macrophages. E) Volcano plot illustrating Trem2 + Cd5l + macrophage marker genes in the proteome from RAW264.7 cells after sh‐ Slc7a5 versus sh‐NC (FC≥1.2 or FC≤0.83, P <0.05). F) Heatmap indicating the fold change in expression of Trem2 + Cd5l + macrophage marker genes in the proteomics of RAW264.7 cells with Slc7a5 knockdown or overexpression. Numbers represent fold change compared to the respective control group ( n = 3). G) Correlation analyses of SLC7A5 and CD5L mRNA levels in human carotid atherosclerotic plaques ( GSE43292 ; n = 64). H) Immunofluorescent staining of BODIPY 493/503 , CD68, and CD5L in aortic sinus from 16‐week HFD‐fed Slc7a5 fl/fl ; Apoe −/− mice and Slc7a5 fl/fl Lyz2 ‐cre; Apoe −/− mice, and quantification of CD68 + CD5L + cells and BODIPY 493/503 area ( n = 9‐10 mice per group). Scale: 200 µm and 50 µm (high magnification). I) Correlation analysis between the proportion of CD68 + CD5L + cells and BODIPY 493/503 area in indicated two groups of mice ( Slc7a5 fl/fl ; Apoe −/− and Slc7a5 fl/fl Lyz2 ‐cre; Apoe −/− ) ( n = 19 mice). J) Flow cytometry detection and quantification of CD5L expression in BCH or JPH203‐treated RAW264.7 cells ( n = 4). K) Quantitative RT‐PCR analysis of Cd5l expression in RAW264.7 cells treated with ox‐LDL with or without mito‐TEMPO for 24 h in Leu‐N or Leu‐D media ( n = 3/group). L) Immunofluorescent staining of CD5L and MitoTracker‐deep red ( n ≥100 cells/group). Scale: 20 µm. M) Pearson's correlation of mean fluorescence intensity (MFI) of CD5L and MitoTracker in RAW264.7 cells treated with or without mito‐TEMPO for 24 h ( n >200 cells). N) Least‐squares means of maximum carotid IMT in UK Biobank participants stratified by high versus low plasma CD5L levels ( n = 2284/group). Data are mean ± SEM. ns, not significant, * P <0.05, ** P <0.01, *** P <0.001, unpaired Student's t ‐test (A, C, H and J), Pearson's correlation test (B, G, I and M), one‐way ANOVA with Dunnett correction (K), Mann‐Whitney test (L), and t ‐test between least squares means (N).

Article Snippet: Breeding strategy: Slc7a5 flox/flox × Lyz2 Cre +/− mice (obtained from the Cyagen Biosciences) → Lyz2 Cre +/− ; Slc7a5 flox/flox ; then crossed with Apoe −/− to obtain Slc7a5 flox/flox Apoe −/− mice.

Techniques: Immunofluorescence, Staining, Marker, Expressing, Knockdown, Over Expression, Control, Flow Cytometry, Quantitative RT-PCR, Fluorescence, Clinical Proteomics, MANN-WHITNEY

Journal: bioRxiv

Article Title: SLCs contribute to endocrine resistance in breast cancer: role of SLC7A5 (LAT1)

doi: 10.1101/555342

Figure Lengend Snippet: Autophagy initiates but does not complete with LAT1 inhibition. A) markers for autophagy and the unfolded protein response show autophagic flux. B) knockdown of GRP78 shows an increase of LAT1 protein after 72 hours. C) The mRNA levels of LAT1 increase with GRP78 knockdown (one replicate is stronger than the other).

Article Snippet: SLC7A5 siRNA and plasmid DNA were obtained from OriGene.

Techniques: Inhibition

Journal: Frontiers in Oncology

Article Title: Genome-wide association studies identify miRNA-194 as a prognostic biomarker for gastrointestinal cancer by targeting ATP6V1F, PPP1R14B, BTF3L4 and SLC7A5

doi: 10.3389/fonc.2022.1025594

Figure Lengend Snippet: The workflow of entire study. First, with the aid of global microRNA expression analysis, a total of 9 prognostic miRNAs for GIC were identified. Second, after analysis of disease-specific survival and progress-free interval, miR-194 turn out to be an optimal prognostic biomarker for GIC. Third, 18 potential target genes greatly downregulated by miR-194 were screened using transcriptome sequencing. Forth, genome-wide CRISPR-CAS9 knockout screening identified 4 miR-194 target genes required for cell proliferation. Fifth, the Ago-HITS-CLIP-seq, qRT-PCR, immunoblotting and cell proliferation assays together confirmed miR-194 inhibited GIC proliferation by targeting ATP6V1F, BTF3L4, PPP1R14B and SLC7A5.

Article Snippet: After blocking with 5% skimmed milk at 4°C for 1h, the membrane was incubated with ATP6V1F antibody (1:1000, Proteintech, 17725-1-AP), BTF3L4 antibody (1:1000, Proteintech, 16500-1-AP), SLC7A5 antibody (1:2000, Abclonal, A2833), PPP1R14B antibody (1:2000, Abclonal, A4677) and β-Actin (1:10000, Proteintech, 20536-1-AP) at 4°C overnight.

Techniques: Expressing, Biomarker Discovery, Sequencing, Genome Wide, CRISPR, Knock-Out, Quantitative RT-PCR, Western Blot

Journal: Frontiers in Oncology

Article Title: Genome-wide association studies identify miRNA-194 as a prognostic biomarker for gastrointestinal cancer by targeting ATP6V1F, PPP1R14B, BTF3L4 and SLC7A5

doi: 10.3389/fonc.2022.1025594

Figure Lengend Snippet: Genome-wide CRISPR-cas9 knockout screening identifies 4 genes required for cell proliferation of GIC cell lines. (A-C) In 18 genes greatly downregulated by miR-194, 4 genes, including ATP6V1F, PPP1R14B, SLC7A5 and BTF3L4, were required for cell proliferation in 26 gastric cancer cell lines, 21 hepatology cancer cell lines and 44 colorectal cancer cell lines. (D-F) Gene expression analysis showed that ATP6V1F, PPP1R14B, SLC7A5 and BTF3L4 were overexpressed in gastric cancer (STAD), colorectal cancer (COADREAD) and liver cancer (LIHC). **P < 0.01. ***P < 0.001.

Article Snippet: After blocking with 5% skimmed milk at 4°C for 1h, the membrane was incubated with ATP6V1F antibody (1:1000, Proteintech, 17725-1-AP), BTF3L4 antibody (1:1000, Proteintech, 16500-1-AP), SLC7A5 antibody (1:2000, Abclonal, A2833), PPP1R14B antibody (1:2000, Abclonal, A4677) and β-Actin (1:10000, Proteintech, 20536-1-AP) at 4°C overnight.

Techniques: Genome Wide, CRISPR, Knock-Out, Gene Expression

Journal: Frontiers in Oncology

Article Title: Genome-wide association studies identify miRNA-194 as a prognostic biomarker for gastrointestinal cancer by targeting ATP6V1F, PPP1R14B, BTF3L4 and SLC7A5

doi: 10.3389/fonc.2022.1025594

Figure Lengend Snippet: The predicted binding sites of miR-194-5p were analyzed in the 18 potential target genes greatly downregulated by miR-194 using the miRcode online web tool.

Article Snippet: After blocking with 5% skimmed milk at 4°C for 1h, the membrane was incubated with ATP6V1F antibody (1:1000, Proteintech, 17725-1-AP), BTF3L4 antibody (1:1000, Proteintech, 16500-1-AP), SLC7A5 antibody (1:2000, Abclonal, A2833), PPP1R14B antibody (1:2000, Abclonal, A4677) and β-Actin (1:10000, Proteintech, 20536-1-AP) at 4°C overnight.

Techniques: Binding Assay

Journal: Frontiers in Oncology

Article Title: Genome-wide association studies identify miRNA-194 as a prognostic biomarker for gastrointestinal cancer by targeting ATP6V1F, PPP1R14B, BTF3L4 and SLC7A5

doi: 10.3389/fonc.2022.1025594

Figure Lengend Snippet: Ago-HITS-CLIP-seq analysis identified ATP6V1F, PPP1R14B, SLC7A5 and BTF3L4 as target genes of miR-194. (A-D) An obvious binding peak of miR-194 was present within the mRNA of ATP6V1F, PPP1R14B, SLC7A5 and BTF3L4, respectively. The statistical results were shown on the right panel. The binding site of miR-194 and PPP1R14B gene is located in in the coding region. While the binding site of miR-194 and other three genes is located in the 3’ untranslated region. (E-H) There was an obvious miR-194 seed sequence binding site inside the miR-194 binding peaks identified by Ago-HITS-CLIP-seq analysis. The binding sites of miR-194 in 4 genes identified by Ago-HITS-CLIP-seq analysis are highly consistent with the predicted results of bioinformatics analysis.

Article Snippet: After blocking with 5% skimmed milk at 4°C for 1h, the membrane was incubated with ATP6V1F antibody (1:1000, Proteintech, 17725-1-AP), BTF3L4 antibody (1:1000, Proteintech, 16500-1-AP), SLC7A5 antibody (1:2000, Abclonal, A2833), PPP1R14B antibody (1:2000, Abclonal, A4677) and β-Actin (1:10000, Proteintech, 20536-1-AP) at 4°C overnight.

Techniques: Binding Assay, Sequencing

Journal: Frontiers in Oncology

Article Title: Genome-wide association studies identify miRNA-194 as a prognostic biomarker for gastrointestinal cancer by targeting ATP6V1F, PPP1R14B, BTF3L4 and SLC7A5

doi: 10.3389/fonc.2022.1025594

Figure Lengend Snippet: MiR-194 inhibited GIC cell proliferation by targeting ATP6V1F, PPP1R14B, SLC7A5 and BTF3L4. (A-C) The expression of miR-194 was negatively associated with the expression of ATP6V1F, PPP1R14B, SLC7A5 and BTF3L4 in gastric cancer (STAD), colorectal cancer (COADREAD) and liver cancer (LIHC). (D) The expression of ATP6V1F, PPP1R14B, SLC7A5 and BTF3L4 were determined after overexpression of miR-194 in GIC cell lines. (E) Overexpression of miR-194 greatly downregulated the protein level of ATP6V1F, PPP1R14B, SLC7A5 and BTF3L4. (F-H) Cell proliferation assays showed that overexpression of miR-194 had similar effects on cell proliferation as siRNA pools containing siRNAs targeting ATP6V1F, PPP1R14B, SLC7A5, and BTF3L4. **P < 0.01.

Article Snippet: After blocking with 5% skimmed milk at 4°C for 1h, the membrane was incubated with ATP6V1F antibody (1:1000, Proteintech, 17725-1-AP), BTF3L4 antibody (1:1000, Proteintech, 16500-1-AP), SLC7A5 antibody (1:2000, Abclonal, A2833), PPP1R14B antibody (1:2000, Abclonal, A4677) and β-Actin (1:10000, Proteintech, 20536-1-AP) at 4°C overnight.

Techniques: Expressing, Over Expression