Journal: Kidney international

Article Title: Over-expression of renal LAT1 and LAT2 and enhanced L-DOPA uptake in SHR immortalized renal proximal tubular cells.

doi: 10.1111/j.1523-1755.2004.00722.x

Figure Lengend Snippet: Fig. 9. Detection of (A) LAT1 and (B) LAT2 in total RNA extracted from WKY and SHR cells using LAT1 and LAT2 rat specific primers.

Article Snippet: Blots were then incubated with rabbit anti-LAT1 polyclonal antibody (1:500; Serotec, Oxford, UK) and rabbit antimouse LAT2 polyclonal antibody [18] (1:2000; kindly provided by Prof. François Verrey) in 5% nonfat dry milk in TBST for 1.5 hours at room temperature.

Techniques:

Journal: Kidney international

Article Title: Over-expression of renal LAT1 and LAT2 and enhanced L-DOPA uptake in SHR immortalized renal proximal tubular cells.

doi: 10.1111/j.1523-1755.2004.00722.x

Figure Lengend Snippet: Fig. 10. Abundance of (A) LAT1 and (B) LAT2 in WKY and SHR cells. Each lane contains equal amount of protein (40 lg). Western blots were repeated 4 to 6 times. Columns indicate relative density and represent the mean of 4 to 6 separate experiments; vertical lines indicate SEM. Significantly different from values for WKY cells. ∗P < 0.05.

Article Snippet: Blots were then incubated with rabbit anti-LAT1 polyclonal antibody (1:500; Serotec, Oxford, UK) and rabbit antimouse LAT2 polyclonal antibody [18] (1:2000; kindly provided by Prof. François Verrey) in 5% nonfat dry milk in TBST for 1.5 hours at room temperature.

Techniques: Western Blot

Journal: Oncotarget

Article Title: SLC7A5 act as a potential leukemic transformation target gene in myelodysplastic syndrome

doi: 10.18632/oncotarget.6512

Figure Lengend Snippet: Prediction of target gene of 7 miRNAs screened by miRNAmicroassay

Article Snippet: The antibodies, used for Western blot analysis, included rabbit polyclonal anti-SLC7A5 antibody (Cell signaling tech Inc., 1:1000), anti-GAPDH antibody (Kangchen Inc., 1:10000), and HRP-conjugated anti-rabbit secondary antibody (Kangchen Inc., 1:10000).

Techniques:

Journal: Oncotarget

Article Title: SLC7A5 act as a potential leukemic transformation target gene in myelodysplastic syndrome

doi: 10.18632/oncotarget.6512

Figure Lengend Snippet: The protein expression was further confirmed by Western blot analysis after three days of transfection. The levels of the SLC7A5 protein were significantly lower in SLC7A5-siRNA group than in the negative control group and Mock. In comparison, GAPDH protein did not vary markedly among the three groups.

Article Snippet: The antibodies, used for Western blot analysis, included rabbit polyclonal anti-SLC7A5 antibody (Cell signaling tech Inc., 1:1000), anti-GAPDH antibody (Kangchen Inc., 1:10000), and HRP-conjugated anti-rabbit secondary antibody (Kangchen Inc., 1:10000).

Techniques: Expressing, Western Blot, Transfection, Negative Control, Comparison

Journal: Oncotarget

Article Title: SLC7A5 act as a potential leukemic transformation target gene in myelodysplastic syndrome

doi: 10.18632/oncotarget.6512

Figure Lengend Snippet: Cell proliferation of SKM-1 cells using CCK-8 assays. The growth of SKM-1 cells in SLC7A5-siRNA group was significantly inhibited compared with the negative control (NC) group ( P <0.05).

Article Snippet: The antibodies, used for Western blot analysis, included rabbit polyclonal anti-SLC7A5 antibody (Cell signaling tech Inc., 1:1000), anti-GAPDH antibody (Kangchen Inc., 1:10000), and HRP-conjugated anti-rabbit secondary antibody (Kangchen Inc., 1:10000).

Techniques: CCK-8 Assay, Negative Control

Journal: Oncotarget

Article Title: SLC7A5 act as a potential leukemic transformation target gene in myelodysplastic syndrome

doi: 10.18632/oncotarget.6512

Figure Lengend Snippet: Cell apoptosis analysis of SLC7A5-siRNA group and negative control group cells was analyzed by flow cytometry. A. Representative histograms of annexin V/PI double-staining flow cytometry. B. Percent apoptosis (including early and late apoptotic cells) determined by flow cytometry (n = 3). * P <0.05 vs control.

Article Snippet: The antibodies, used for Western blot analysis, included rabbit polyclonal anti-SLC7A5 antibody (Cell signaling tech Inc., 1:1000), anti-GAPDH antibody (Kangchen Inc., 1:10000), and HRP-conjugated anti-rabbit secondary antibody (Kangchen Inc., 1:10000).

Techniques: Negative Control, Flow Cytometry, Double Staining, Control

Journal: Oncotarget

Article Title: SLC7A5 act as a potential leukemic transformation target gene in myelodysplastic syndrome

doi: 10.18632/oncotarget.6512

Figure Lengend Snippet: Cell cycle assay by flow cytometry in SLC7A5-siRNAand negative control group

Article Snippet: The antibodies, used for Western blot analysis, included rabbit polyclonal anti-SLC7A5 antibody (Cell signaling tech Inc., 1:1000), anti-GAPDH antibody (Kangchen Inc., 1:10000), and HRP-conjugated anti-rabbit secondary antibody (Kangchen Inc., 1:10000).

Techniques: Cell Cycle Assay, Flow Cytometry, Negative Control

Journal: Oncotarget

Article Title: SLC7A5 act as a potential leukemic transformation target gene in myelodysplastic syndrome

doi: 10.18632/oncotarget.6512

Figure Lengend Snippet: Cell cycle distribution of SLC7A5-siRNA group and negative control group cells was analyzed by flow cytometry with PI staining after 48 h treatment. A. Representative flow cytometry histograms. B. Cell distribution at G0/G1, S, and G2/M phases of the cell cycle (n = 3). * P <0.05 vs control.

Article Snippet: The antibodies, used for Western blot analysis, included rabbit polyclonal anti-SLC7A5 antibody (Cell signaling tech Inc., 1:1000), anti-GAPDH antibody (Kangchen Inc., 1:10000), and HRP-conjugated anti-rabbit secondary antibody (Kangchen Inc., 1:10000).

Techniques: Negative Control, Flow Cytometry, Staining, Control

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Amino acid transporter LAT1 (SLC7A5) promotes metabolic rewiring in TNBC progression through the L-Trp/QPRT/NAD + pathway

doi: 10.1186/s13046-025-03446-z

Figure Lengend Snippet: LAT1 is upregulated in TNBC and associated with poor prognosis. A A pan-cancer analysis of LAT1 mRNA expression across various cancer types and their corresponding normal tissues using the TCGA database. B-E LAT1 mRNA levels and clinical data for breast cancer (BC) were retrieved from the TCGA database. B LAT1 levels in normal breast tissues and BC tissues. C LAT1 levels in three subtypes of BC—luminal, HER2 +, and TNBC—compared to normal breast tissues. D Kaplan–Meier plot of overall survival (OS) in BC patients with high or low LAT1 expression. Survival curves were compared using the log-rank test. E Kaplan–Meier analysis of OS in the basal subtype of BC with high or low LAT1 expression. Survival curves were compared using the log-rank test. F Human breast cancer tissue microarrays were stained for LAT1 protein expression using IHC in normal breast tissues and three subtypes of BC, including luminal, HER2 +, and TNBC. An IHC staining score for LAT1 was calculated for each sample based on both the staining intensity and the percentage of positive cells. Representative images are shown. Scale bar = 600 µm (Top); 300 µm (Inset). G Quantification of the IHC staining for LAT1. H A Kaplan–Meier plot of OS (left) and disease-free survival (DFS, right) was generated for BC patients with high or low LAT1 expression, stratified based on IHC staining scores. Survival curves were compared using the log-rank test. I The correlation analysis between LAT1 expressions and prognosis of TNBC patients. NED: No evidence of disease. ED: Evidence of disease. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The primary antibodies used in this study include: LAT1 Rabbit Antibody (1:1000, #5347, Cell Signaling Technology, USA); Phospho-PKM2 (Tyr105) Rabbit Antibody (1:1000, #3827, Cell Signaling); PKM2 Rabbit Antibody (1:1000, #4053, Cell Signaling); LDHA Rabbit Antibody (1:1000, #3582, Cell Signaling); Phospho-LDHA (Tyr10) Rabbit Antibody (1:1000, #PA5-105445, Thermo Scientific); QPRT Rabbit Antibody (1:1000, #25174-1-AP, Protein Tech., USA); β-actin Mouse Antibody (1:2000, #sc-47778, Santa Cruz Biotechnology, USA).

Techniques: Expressing, Staining, Immunohistochemistry, Generated

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Amino acid transporter LAT1 (SLC7A5) promotes metabolic rewiring in TNBC progression through the L-Trp/QPRT/NAD + pathway

doi: 10.1186/s13046-025-03446-z

Figure Lengend Snippet: Inhibition of LAT1 suppresses TNBC cell viability, proliferation, migration, and in vivo tumor growth. A-F Cells were transiently transfected with 100 nM Smartpool LAT1 siRNA or a negative siRNA control (NC) for 72 h before the downstream assays. A MTT assay was performed to assess cell viability. B A colorimetric BrdU incorporation assay was performed to evaluate cell proliferation. C Immunofluorescent staining for BrdU was performed to assess cell proliferation. Left: Representative images. Right: Percentage of BrdU-positive cells. D Cell apoptosis was measured using Annexin V-FITC and PI double staining with flow cytometry. Left: Representative images. Right: Fold change in the rate of apoptotic cells. E A transwell migration assay was performed to assess cell migration ability. Left: Representative images. Right: Fold change in the number of migrated cells. Scale bar = 200 µm. F A transwell in-vitro invasion assay was conducted to assess cell invasion ability. Left: Representative images. Right: Fold change in the number of invading cells. Scale bar = 200 µm. G An orthotopic breast cancer model was established in nude mice using MDA-MB-231 control cells or LAT1 knockdown cells. The tumor images and growth curves were shown. N = 10 per group. Scale bar = 1 cm H A syngeneic breast cancer model was established in C57BL/6 mice using PY8119 murine TNBC cells. The mice were treated with either vehicle control or 15 mg/kg of JPH203 via subcutaneous injection five times per week for two weeks. Left: Representative images of tumors. Middle: Tumor growth curve. Right: In vivo bioluminescence imaging of the tumors. Three independent experiments were performed with a minimum of three biological replicates. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The primary antibodies used in this study include: LAT1 Rabbit Antibody (1:1000, #5347, Cell Signaling Technology, USA); Phospho-PKM2 (Tyr105) Rabbit Antibody (1:1000, #3827, Cell Signaling); PKM2 Rabbit Antibody (1:1000, #4053, Cell Signaling); LDHA Rabbit Antibody (1:1000, #3582, Cell Signaling); Phospho-LDHA (Tyr10) Rabbit Antibody (1:1000, #PA5-105445, Thermo Scientific); QPRT Rabbit Antibody (1:1000, #25174-1-AP, Protein Tech., USA); β-actin Mouse Antibody (1:2000, #sc-47778, Santa Cruz Biotechnology, USA).

Techniques: Inhibition, Migration, In Vivo, Transfection, Control, MTT Assay, BrdU Incorporation Assay, Staining, Double Staining, Flow Cytometry, Transwell Migration Assay, In Vitro, Invasion Assay, Knockdown, Injection, Imaging

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Amino acid transporter LAT1 (SLC7A5) promotes metabolic rewiring in TNBC progression through the L-Trp/QPRT/NAD + pathway

doi: 10.1186/s13046-025-03446-z

Figure Lengend Snippet: LAT1 silencing rewires TNBC cell metabolism by reducing glycolysis. A Differentially expressed genes were identified between BC patients with high and low LAT1 expression levels using TCGA data. GSEA was then performed on differential genes to identify enriched hallmark pathways. B-C Cells were transiently transfected with 100 nM Smartpool siLAT1 or a negative siRNA control (NC) for 72 h. The relative levels of cytosolic B pyruvate and C lactate were measured using the Pyruvate Assay Kit and Lactate Assay Kit, respectively, according to the manufacturer’s instructions. D-F Metabolites were profiled in shNC and shLAT1 MDA-MB-231 cells using an LC-MS-based assay. D Metabolite enrichment analysis was performed to analyze the differentially expressed metabolites using MetaboAnalyst software. The bar chart shows the enriched metabolic pathways in the differential metabolites. E The levels of G-6-P, and lactic acid in shNC and shLAT1 MDA-MB-231 cells were determined by the metabolomics assay. Peak intensities were normalized for comparison ( n = 4). F MDA-MB-231 cells were treated with vehicle control or 10 µM JPH203 for 10 min. The levels of G-6-P, and lactic acid were measured by LC-MS assay. Peak intensities were normalized for comparison ( n = 4). G A Seahorse assay was conducted to measure ECAR in shNC or shLAT1 MDA-MB-231 cells ( n = 3). Three independent experiments were performed with a minimum of three biological replicates. * P < 0.05; ** P < 0.01; *** P < 0.001; ns. not significant

Article Snippet: The primary antibodies used in this study include: LAT1 Rabbit Antibody (1:1000, #5347, Cell Signaling Technology, USA); Phospho-PKM2 (Tyr105) Rabbit Antibody (1:1000, #3827, Cell Signaling); PKM2 Rabbit Antibody (1:1000, #4053, Cell Signaling); LDHA Rabbit Antibody (1:1000, #3582, Cell Signaling); Phospho-LDHA (Tyr10) Rabbit Antibody (1:1000, #PA5-105445, Thermo Scientific); QPRT Rabbit Antibody (1:1000, #25174-1-AP, Protein Tech., USA); β-actin Mouse Antibody (1:2000, #sc-47778, Santa Cruz Biotechnology, USA).

Techniques: Expressing, Transfection, Control, Pyruvate Assay, Lactate Assay, Liquid Chromatography with Mass Spectroscopy, Software, Comparison

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Amino acid transporter LAT1 (SLC7A5) promotes metabolic rewiring in TNBC progression through the L-Trp/QPRT/NAD + pathway

doi: 10.1186/s13046-025-03446-z

Figure Lengend Snippet: LAT1 enhances glycolytic activities via activating PKM2 and LDHA. A LAT1 and PKM2 expression levels in BC were retrieved from TCGA. The correlation between LAT1 and PKM2 expression was assessed using Spearman's rank correlation analysis. B Kaplan–Meier analysis for the OS in patients with high or low PKM2 expression levels in the basal BC subtype using TCGA data. C-E The levels of p-PKM2 (Tyr105) protein in normal and BC tissues were evaluated by IHC using a human breast cancer tissue microarray. An IHC staining score was calculated for p-PKM2. C Representative images are shown. Scale bar = 600 µm (Top); 300 µm (Inset). D Quantification of IHC staining for p-PKM2. E Kaplan–Meier plot of the OS and DFS in BC patients with high or low expression of p-PKM2 (Try105) based on tissue microarray IHC staining score. F LAT1 and LDHA expression levels in BC were retrieved from TCGA. The correlation between LAT1 and LDHA expression was assessed using Spearman's rank correlation analysis. G Kaplan–Meier plot of OS in the basal subtype of BC patients with high or low LDHA expression levels, using TCGA data. Survival curves were compared using the log-rank test. H-J Human breast cancer tissue microarrays were stained for p-LDHA (Tyr10) levels by IHC. H Representative images were shown. Scale bar = 600 µm (Top); 300 µm (Inset). I Quantification of IHC staining for p-LDHA. J Kaplan–Meier survival analysis of OS and DFS in BC patients with high or low p-LDHA (Tyr10) expression based on IHC staining scores. K The protein levels of LAT1, PKM2, p-PKM2, LDHA, and p-LDHA were measured by Western blotting in TNBC cells with transient or stable LAT1 knockdown. L MDA-MB231 cells were treated with JPH203 at different time points and doses as indicated. The protein levels of LAT1, PKM2, p-PKM2, LDHA, and p-LDHA were measured by immunoblotting. Three independent experiments were performed with a minimum of three biological replicates. ** P < 0.01; *** P < 0.001

Article Snippet: The primary antibodies used in this study include: LAT1 Rabbit Antibody (1:1000, #5347, Cell Signaling Technology, USA); Phospho-PKM2 (Tyr105) Rabbit Antibody (1:1000, #3827, Cell Signaling); PKM2 Rabbit Antibody (1:1000, #4053, Cell Signaling); LDHA Rabbit Antibody (1:1000, #3582, Cell Signaling); Phospho-LDHA (Tyr10) Rabbit Antibody (1:1000, #PA5-105445, Thermo Scientific); QPRT Rabbit Antibody (1:1000, #25174-1-AP, Protein Tech., USA); β-actin Mouse Antibody (1:2000, #sc-47778, Santa Cruz Biotechnology, USA).

Techniques: Expressing, Microarray, Immunohistochemistry, Staining, Western Blot, Knockdown

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Amino acid transporter LAT1 (SLC7A5) promotes metabolic rewiring in TNBC progression through the L-Trp/QPRT/NAD + pathway

doi: 10.1186/s13046-025-03446-z

Figure Lengend Snippet: LAT1 promotes NAD + de novo synthesis through L-Trp/QPRT for glycolysis. A-C MDA-MB-231 cells were transfected with 100 nM LAT1 Smartpool siRNA or treated with 10 µM JPH203 for 10 min to inhibit the activity of LAT1. LC–MS-based metabolomics was performed to profile changes in cellular metabolites. The relative levels of intracellular ( A ) L-Tryptophan, B NAD + , and C NADH in LAT1-inhibited and control cells were measured by the metabolomics assay. Peak intensities were normalized for comparison ( n = 4). D TNBC cells were transfected with 100 nM LAT1 Smartpool siRNA or control siRNA (NC) for 72 h. The relative cytosolic NAD + /NADH ratios were measured using a NAD + /NADH Quantification Kit. E TNBC cells were treated with 4 mM L-Tryptophan for 30 min. The relative cytosolic NAD +/NADH ratios were determined as described above. F MDA-MB-231 cells were transiently transfected with 100 nM Smartpool siLAT1 or a negative siRNA control (NC) for 72 h. The cells were then treated with either vehicle control or 4.0 mM NAD⁺ for 20 min. Pyruvate and lactate levels were measured using the corresponding assay kits. G MDA-MB-231 cells were treated with vehicle control, 4.0 mM NAD + , 10 µM JPH203, or a combination 4.0 mM NAD + and 10 µM JPH203 for 10 min. Pyruvate and lactate levels were measured using the corresponding assay kits. H The correlation between LAT1 and QPRT in BC was evaluated using Spearman's rank correlation analysis of TCGA data. I QPRT expression and survival data were obtained from the TCGA database. Kaplan–Meier survival curves for OS were plotted for patients with high or low QPRT expression, both for overall BC patients (left) and for those with the basal subtype of BC (right). J LAT1 was silenced in TNBC cells using either siRNA transfection or lentiviral shRNA transduction. QPRT protein expression was assessed by immunoblotting. K MDA-MB-231 and MDA-MB-468 cells were incubated in amino acid–free medium supplemented with L-tryptophan for 30 min. The protein levels of QPRT, PKM2, p-PKM2, LDHA, and p-LDHA were evaluated by Western blotting. L MDA-MB-231 and BT549 cells were transiently transfected with 100 nM SmartPool siLAT1 or control siRNA (NC) for 72 h, followed by incubation in amino acid–free medium supplemented with L-tryptophan for 30 min. The protein levels of QPRT, PKM2, p-PKM2, LDHA, and p-LDHA were analyzed by immunoblotting. M Cells were treated with NAD + for 20 min. The protein levels of PKM2, p-PKM2, LDHA, and p-LDHA were measured by immunoblotting. * P < 0.05; ** P < 0.01; *** P < 0.001. Three independent experiments were performed with a minimum of three biological replicates

Article Snippet: The primary antibodies used in this study include: LAT1 Rabbit Antibody (1:1000, #5347, Cell Signaling Technology, USA); Phospho-PKM2 (Tyr105) Rabbit Antibody (1:1000, #3827, Cell Signaling); PKM2 Rabbit Antibody (1:1000, #4053, Cell Signaling); LDHA Rabbit Antibody (1:1000, #3582, Cell Signaling); Phospho-LDHA (Tyr10) Rabbit Antibody (1:1000, #PA5-105445, Thermo Scientific); QPRT Rabbit Antibody (1:1000, #25174-1-AP, Protein Tech., USA); β-actin Mouse Antibody (1:2000, #sc-47778, Santa Cruz Biotechnology, USA).

Techniques: Transfection, Activity Assay, Liquid Chromatography with Mass Spectroscopy, Control, Comparison, Expressing, shRNA, Transduction, Western Blot, Incubation

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Amino acid transporter LAT1 (SLC7A5) promotes metabolic rewiring in TNBC progression through the L-Trp/QPRT/NAD + pathway

doi: 10.1186/s13046-025-03446-z

Figure Lengend Snippet: QPRT mediated LAT1-induced upregulation of p-PKM2 and p-LDHA. A-B MDA-MB-231 and MDA-MB-468 cells were transfected with 100 nM SmartPool QPRT siRNA or a negative control siRNA (NC) for 72 h. A The relative cytosolic NAD +/NADH ratios were measured using a NAD/NADH Quantification Kit. B The protein levels of QPRT, PKM2, p-PKM2, LDHA, and p-LDHA were measured by Western blotting. C-F The shLAT1-expressing MDA-MB-231 cells were transfected with a QPRT-expressing plasmid or a vector control. C The protein levels of LAT1, QPRT, PKM2, p-PKM2, LDHA, and p-LDHA were measured by immunoblotting. D Relative cytosolic NAD +/NADH ratios were measured. E Pyruvate and lactate productions were assessed using pyruvate and lactate assay kits, respectively. F Cell proliferation assay was performed using BrdU assay kits. G A graphic summary of the proposed LAT1/QPRT/PKM2/LDHA signaling pathway. Three independent experiments were performed with a minimum of three biological replicates. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The primary antibodies used in this study include: LAT1 Rabbit Antibody (1:1000, #5347, Cell Signaling Technology, USA); Phospho-PKM2 (Tyr105) Rabbit Antibody (1:1000, #3827, Cell Signaling); PKM2 Rabbit Antibody (1:1000, #4053, Cell Signaling); LDHA Rabbit Antibody (1:1000, #3582, Cell Signaling); Phospho-LDHA (Tyr10) Rabbit Antibody (1:1000, #PA5-105445, Thermo Scientific); QPRT Rabbit Antibody (1:1000, #25174-1-AP, Protein Tech., USA); β-actin Mouse Antibody (1:2000, #sc-47778, Santa Cruz Biotechnology, USA).

Techniques: Transfection, Negative Control, Western Blot, Expressing, Plasmid Preparation, Control, Lactate Assay, Proliferation Assay, BrdU Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Amino acid transporter LAT1 (SLC7A5) promotes metabolic rewiring in TNBC progression through the L-Trp/QPRT/NAD + pathway

doi: 10.1186/s13046-025-03446-z

Figure Lengend Snippet: Knockdown of LAT1 sensitized resistant TNBC cells to Doxorubicin. A A schematic illustration of the establishment of an acquired Dox-resistant PDXmouse model. The PDX tumor without Dox treatment was set up as a control. After four passages, both the parental and resistant PDX tumors were harvested, and primary cells were dissociated using enzymatic digestion. B The dissociated primary cells were seeded onto a 96-well plate and treated with different concentrations of Dox for 72 h. The IC 50 values of Dox in the cells were determined using an MTT assay. C Parental and Dox-resistant MDA-MB-231 cells were treated with different concentrations of Dox for 72 h. The IC 50 values were determined using an MTT assay. D The MDA-MB-231-R cells were transfected with 100 nM siLAT1 or siNC for 24 h. The cells were then treated with vehicle control, 200 nM, or 400 nM Dox for 48 h followed by apoptosis assay. E MDA-MB-231-R stably LAT1 knockdown cells (shLAT1) and control cells (shNC) were treated with Dox at different doses. The cell viability was measured using the colorimetric BrdU incorporation assay. F The protein levels of LAT1, QPRT, PKM2, p-PKM2, LDHA, and p-LDHA were measured by immunoblotting in sensitive and Dox-resistant MDA-MB-231 cells, as well as in primary cells dissociated from parental and Dox-resistant PDX tumors. G MDA-MB-231-R cells were treated with a vehicle control, 10 mM 2-DG, or 5 µM JPH203 in combination with varying concentrations of Dox for 72 h. IC₅₀ values were determined using an MTT assay. H shLAT1-expressing MDA-MB-231-R cells were treated with vehicle control, 1 mM L-Trp, or 1 mM NAD⁺, along with varying concentrations of Dox for 72 h. The IC₅₀ values of Dox in the cells were then determined using the MTT assay. I The shLAT1-expressing MDA-MB-231 cells were transfected with either a QPRT-expressing plasmid or a vector control, followed by incubation with varying concentrations of Dox for 72 h. The IC₅₀ values of Dox in the cells were then determined using the MTT assay. J MDA-MB-231-R cells were treated with Dox alone, JPH203 alone, or a combination of both drugs at the indicated doses for 72 h. Cell viability was assessed using MTT assays, and combination index (CI) analysis was performed. The figure illustrating the combined effects was generated using CompuSyn software. Three independent experiments were performed with a minimum of three biological replicates.* P < 0.05; *** P < 0.001

Article Snippet: The primary antibodies used in this study include: LAT1 Rabbit Antibody (1:1000, #5347, Cell Signaling Technology, USA); Phospho-PKM2 (Tyr105) Rabbit Antibody (1:1000, #3827, Cell Signaling); PKM2 Rabbit Antibody (1:1000, #4053, Cell Signaling); LDHA Rabbit Antibody (1:1000, #3582, Cell Signaling); Phospho-LDHA (Tyr10) Rabbit Antibody (1:1000, #PA5-105445, Thermo Scientific); QPRT Rabbit Antibody (1:1000, #25174-1-AP, Protein Tech., USA); β-actin Mouse Antibody (1:2000, #sc-47778, Santa Cruz Biotechnology, USA).

Techniques: Knockdown, Control, MTT Assay, Transfection, Apoptosis Assay, Stable Transfection, BrdU Incorporation Assay, Western Blot, Expressing, Plasmid Preparation, Incubation, Generated, Software

Journal: Nuclear medicine and biology

Article Title: Enhancing the accumulation level of 3-[ 18 F]fluoro-L-α-methyltyrosine in tumors by preloading probenecid.

doi: 10.1016/j.nucmedbio.2021.11.006

Figure Lengend Snippet: Fig. 5. Analysis of the effect of probenecid on LS180 cell. (A) LAT1 expression and (B) cellular uptake (mean ± SD, n ≥4) of LS180 cell treated with or without probenecid (1 mM). n.s., not significant compared with control group (without probenecid).

Article Snippet: Then, blots were incubated with mouse anti-LAT1 antibody (sc374232, Santa Cruz Biotechnology, Dallas, TX) or rabbit anti-β-actin antibody (#4970, Cell Signaling Technology, Danvers, MA) at 4 °C overnight.

Techniques: Expressing, Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Overexpression of non-functional LAT1/4F2hc in renal proximal tubular epithelial cells from the spontaneous hypertensive rat.

doi: 10.1159/000107537

Figure Lengend Snippet: Fig. 1. Abundance of (A) LAT1 and (B) 4F2hc in WKY and SHR freshly isolated renal proximal tubules and WKY and SHR immortalized renal proximal tubular cells. Columns indicate density and represent the mean of 4-6 separate experiments; vertical lines indicate S.E.M. Significantly different from values for WKY cells (* P<0.05). (C) Each lane contains equal amount of protein (40 µg).

Article Snippet: Blots were then incubated with anti-LAT1 rabbit polyclonal antibody (1:1,500; Serotec, Oxford, UK) or the anti-4F2hc rabbit polyclonal antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the anti β-actin primary antibody (1:10,000; Upstate Biotechnologies) in 5% non-fat dry milk in PBS-T overnight at 4oC.

Techniques: Isolation

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Overexpression of non-functional LAT1/4F2hc in renal proximal tubular epithelial cells from the spontaneous hypertensive rat.

doi: 10.1159/000107537

Figure Lengend Snippet: Fig. 11. [14C]-L-leucine (0.25 µM) uptake in LAT1 siRNA- and 4F2hc siRNA- treated WKY and SHR immortalized renal proximal tubular cells, for 24h. Columns represent the mean of 4-8 experiments per group; vertical lines show S.E.M.. Significantly different from control value (* P<0.05).

Article Snippet: Blots were then incubated with anti-LAT1 rabbit polyclonal antibody (1:1,500; Serotec, Oxford, UK) or the anti-4F2hc rabbit polyclonal antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the anti β-actin primary antibody (1:10,000; Upstate Biotechnologies) in 5% non-fat dry milk in PBS-T overnight at 4oC.

Techniques: Control

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Overexpression of non-functional LAT1/4F2hc in renal proximal tubular epithelial cells from the spontaneous hypertensive rat.

doi: 10.1159/000107537

Figure Lengend Snippet: Fig. 12. Abundance of (A) LAT1 and (B) 4F2hc proteins in LAT1 siRNA- and 4F2hc siRNA-treated WKY and SHR immortalized renal proximal tubular cells, for 24h. Columns represent the mean of 4 experiments per group; vertical lines show S.E.M.. Significantly different from control value (* P<0.05). Each lane contains equal amount of protein (40 µg).

Article Snippet: Blots were then incubated with anti-LAT1 rabbit polyclonal antibody (1:1,500; Serotec, Oxford, UK) or the anti-4F2hc rabbit polyclonal antibody (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and the anti β-actin primary antibody (1:10,000; Upstate Biotechnologies) in 5% non-fat dry milk in PBS-T overnight at 4oC.

Techniques: Control

Journal: Scientific Reports

Article Title: DRAM1 enhances the proliferation and metastasis of gastric cancer through the PI3K/AKT/mTOR signaling pathway and energy metabolism

doi: 10.1038/s41598-025-87389-7

Figure Lengend Snippet: DRAM1 mediated energy metabolism in gastric cancer cells through mTOR. ( a ) GO analysis was performed to analyze the differential genes of DRAM1 silencing GC cells and NC GC cells, which were significantly related to energy metabolism pathway. ( b ) KEGG analysis was conducted to examine the differential genes between DRAM1 silenced gastric cancer cells and non-silenced gastric cancer cells. The results revealed a significant association with the energy metabolism pathway. ( c ) KEGG was used to analyze the linkages between the metabolic pathways of differentially enriched genes. ( d ) Knocking down of DRAM1 inhibited the mRNA expression of HK2 (Ctrl vs sh DRAM1 #1,ns; Ctrl vs sh DRAM1 #2, *: P = 0.013), PFK1 (Ctrl vs sh DRAM1 #1, **: P = 0.001; Ctrl vs sh DRAM1 #2, **: P = 0.002), GCK (Ctrl vs sh DRAM1 #1, *: P = 0.011; Ctrl vs sh DRAM1 #2, **: P = 0.004), PKM2 (Ctrl vs sh DRAM1 #1, *: P = 0.042; Ctrl vs sh DRAM1 #2,ns), ATP6V1A (Ctrl vs sh DRAM1 #1, **: P = 0.001; Ctrl vs sh DRAM1 #2, **: P = 0.002), LDHA (Ctrl vs sh DRAM1 #1, **: P = 0.004; Ctrl vs sh DRAM1 #2, **: P = 0.006), c-MYC (Ctrl vs sh DRAM1 #1, ***: P < 0.001; Ctrl vs sh DRAM1 #2, **: P = 0.002) and IDH1 (Ctrl vs sh DRAM1 #1, * : P = 0.038; Ctrl vs sh DRAM1 #2, **: P = 0.005) in gastric cancer cells by q-PCR analysis. ( e ) Western blotting confirmed that knockdown of DRAM1 inhibited the expression of PKM2, HK2, GCK, SLC1A5 and LDHA proteins in gastric cancer cells. ( f ) Western blotting confirmed that overexpression of DRAM1 promoted the expression of PKM2, GCK, SLC1A5, LDHA, PFK1 and SLC7A5 proteins in gastric cancer cells. ( g ) Knockdown of DRAM1 inhibit ATP production in gastric cancer cells (Ctrl vs sh DRAM1 #1, **: P = 0.006; Ctrl vs sh DRAM1 #2, **: P = 0.008). ( h ) Knockdown of DRAM1 inhibited lactate production in gastric cancer cells (Ctrl vs sh DRAM1 #1, **: P = 0.002; Ctrl vs sh DRAM1 #2, **: P = 0.002).

Article Snippet: The following primary antibodies were used: β-actin (proteintech; 66009-1-immunoglobin [Ig]; 1:3000), DRAM1 (Abcam; Ab208160; 1:1000), SLC1A5 (proteintech; 20350-1-AP; 1:2000), SLC7A5 (proteintech; 28670-1-AP; 1:2000), mTOR (proteintech; 66888-1-Ig; 1:2000), p-mTOR (proteintech; 67778-1-Ig; 1:2000), P62 (proteintech; 18420-1-AP; 1:2000), p-AKT (proteintech; 66444-1-Ig; 1:2000), E-cadherin (proteintech; 20874-1-AP; 1:2000), N-cadherin (proteintech; 22018-1-AP; 1:2000), Bax (Cell Signaling Technology; 2772S; 1:1000), Bcl-2 (Cell Signaling Technology; 15071S; 1:1000), PI3K (Wanleibio; WL03380; 1:1000), AKT (Cell Signaling Technology; 9272S; 1:1000), HK2 (Abways; AB3194; 1:1000), PFK1 (Abways; CY8047; 1:1000), GCK (Abways; CY8036; 1:1000), LDHA (bioworld; BS6179;1:1000), PKM2 (Cell Signaling Technology; 4053S; 1:1000), p53 (proteintech; 60283-22-Ig; 1:2000),LC3 (proteintech; 14600-1-AP; 1:2000), anti-rabbit antibody horseradish peroxidase (HRP) rabbit (Cell Signaling Technology; 7074S; 1:3000), and anti-mouse antibody HRP mouse (Cell Signaling Technology; 7076S; 1:3000).

Techniques: Expressing, Western Blot, Knockdown, Over Expression