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mtx  (MedChemExpress)
94
MedChemExpress mtx
a. Simplified metabolism of 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). Asterisk denotes ability of 6-TGMP to be transformed into 6-meTGMP that may inhibit de novo purine synthesis. b. FACS-based growth competition comparing ΔNUDT5 and mutants to wildtype HEK293T cells treated with 6-TG. Data are individual values from n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments. c. Chemical structures of adenosine-5’-monophosphate (AMP) and 6-methylthioinosine-5’-monophosphate (6-meTIMP). d. Left – alignment of molecular glue interface of AMP and 6-meTIMP showing cryo-EM density for the nucleotides. Right – rearrangement of PPAT interface residues in the 6-meTIMP structure (dark sidechains) compared to the AMP-bounds structure (light sidechains) e. Hydrophobic pocket of PPAT engaged by 6-meTIMP. f. 2D-ligand diagram of the 6-meTIMP molecular glue interface. g. PPAT activity assay measuring nucleotide-dependent inhibition in the presence of NUDT5 with 0.25 mM PRPP. Data points are the mean and error bars are SEM from n=3 independent experiments. h. Left – Western blot of endogenous NUDT5 3xFLAG immunoprecipitations following 16-hour treatment <t>with</t> <t>methotrexate</t> (2 µM), 6-MP (50 µM), and <t>MTX</t> + 6-MP. Right – Quantification of PPAT immunoprecipitation normalized to NUDT5 3xFLAG bait and compared to a DMSO-treated control condition. Data are individual values from n=3 independent biological replicate experiments and error bars are SEM. i. Fractional enrichment of AMP (M+2) and GMP (M+3) isotopologs in [ 15 N-amide]-glutamine labeling experiments conducted in the presence of 6-MP. Data points are individual values of n=6 biological replicates from two independent experiments and error bars are SEM. Statistical comparisons were performed using Welch’s two-tailed t-test with Bonferroni correction between wildtype and each mutant. *** denotes a Bonferroni adjusted p-value < 0.001 and ** is p-value < 0.01. j. FACS-based growth competition experiment comparing growth of ΔNUDT5 and endogenous L217A/K218A (LKAA) NUDT5 mutants to wildtype HEK293T treated with 6-MP and 6-TG. Data show n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments.
Mtx, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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folic acid methotrexate  (Micromedex Inc)
86
Micromedex Inc folic acid methotrexate
a. Simplified metabolism of 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). Asterisk denotes ability of 6-TGMP to be transformed into 6-meTGMP that may inhibit de novo purine synthesis. b. FACS-based growth competition comparing ΔNUDT5 and mutants to wildtype HEK293T cells treated with 6-TG. Data are individual values from n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments. c. Chemical structures of adenosine-5’-monophosphate (AMP) and 6-methylthioinosine-5’-monophosphate (6-meTIMP). d. Left – alignment of molecular glue interface of AMP and 6-meTIMP showing cryo-EM density for the nucleotides. Right – rearrangement of PPAT interface residues in the 6-meTIMP structure (dark sidechains) compared to the AMP-bounds structure (light sidechains) e. Hydrophobic pocket of PPAT engaged by 6-meTIMP. f. 2D-ligand diagram of the 6-meTIMP molecular glue interface. g. PPAT activity assay measuring nucleotide-dependent inhibition in the presence of NUDT5 with 0.25 mM PRPP. Data points are the mean and error bars are SEM from n=3 independent experiments. h. Left – Western blot of endogenous NUDT5 3xFLAG immunoprecipitations following 16-hour treatment <t>with</t> <t>methotrexate</t> (2 µM), 6-MP (50 µM), and <t>MTX</t> + 6-MP. Right – Quantification of PPAT immunoprecipitation normalized to NUDT5 3xFLAG bait and compared to a DMSO-treated control condition. Data are individual values from n=3 independent biological replicate experiments and error bars are SEM. i. Fractional enrichment of AMP (M+2) and GMP (M+3) isotopologs in [ 15 N-amide]-glutamine labeling experiments conducted in the presence of 6-MP. Data points are individual values of n=6 biological replicates from two independent experiments and error bars are SEM. Statistical comparisons were performed using Welch’s two-tailed t-test with Bonferroni correction between wildtype and each mutant. *** denotes a Bonferroni adjusted p-value < 0.001 and ** is p-value < 0.01. j. FACS-based growth competition experiment comparing growth of ΔNUDT5 and endogenous L217A/K218A (LKAA) NUDT5 mutants to wildtype HEK293T treated with 6-MP and 6-TG. Data show n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments.
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4 amino n10 methyl folic acid amethopterin  (Lederle Laboratories)
86
Lederle Laboratories 4 amino n10 methyl folic acid amethopterin
a. Simplified metabolism of 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). Asterisk denotes ability of 6-TGMP to be transformed into 6-meTGMP that may inhibit de novo purine synthesis. b. FACS-based growth competition comparing ΔNUDT5 and mutants to wildtype HEK293T cells treated with 6-TG. Data are individual values from n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments. c. Chemical structures of adenosine-5’-monophosphate (AMP) and 6-methylthioinosine-5’-monophosphate (6-meTIMP). d. Left – alignment of molecular glue interface of AMP and 6-meTIMP showing cryo-EM density for the nucleotides. Right – rearrangement of PPAT interface residues in the 6-meTIMP structure (dark sidechains) compared to the AMP-bounds structure (light sidechains) e. Hydrophobic pocket of PPAT engaged by 6-meTIMP. f. 2D-ligand diagram of the 6-meTIMP molecular glue interface. g. PPAT activity assay measuring nucleotide-dependent inhibition in the presence of NUDT5 with 0.25 mM PRPP. Data points are the mean and error bars are SEM from n=3 independent experiments. h. Left – Western blot of endogenous NUDT5 3xFLAG immunoprecipitations following 16-hour treatment <t>with</t> <t>methotrexate</t> (2 µM), 6-MP (50 µM), and <t>MTX</t> + 6-MP. Right – Quantification of PPAT immunoprecipitation normalized to NUDT5 3xFLAG bait and compared to a DMSO-treated control condition. Data are individual values from n=3 independent biological replicate experiments and error bars are SEM. i. Fractional enrichment of AMP (M+2) and GMP (M+3) isotopologs in [ 15 N-amide]-glutamine labeling experiments conducted in the presence of 6-MP. Data points are individual values of n=6 biological replicates from two independent experiments and error bars are SEM. Statistical comparisons were performed using Welch’s two-tailed t-test with Bonferroni correction between wildtype and each mutant. *** denotes a Bonferroni adjusted p-value < 0.001 and ** is p-value < 0.01. j. FACS-based growth competition experiment comparing growth of ΔNUDT5 and endogenous L217A/K218A (LKAA) NUDT5 mutants to wildtype HEK293T treated with 6-MP and 6-TG. Data show n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments.
4 Amino N10 Methyl Folic Acid Amethopterin, supplied by Lederle Laboratories, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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methotrexate  (MedChemExpress)
94
MedChemExpress methotrexate
a. Simplified metabolism of 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). Asterisk denotes ability of 6-TGMP to be transformed into 6-meTGMP that may inhibit de novo purine synthesis. b. FACS-based growth competition comparing ΔNUDT5 and mutants to wildtype HEK293T cells treated with 6-TG. Data are individual values from n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments. c. Chemical structures of adenosine-5’-monophosphate (AMP) and 6-methylthioinosine-5’-monophosphate (6-meTIMP). d. Left – alignment of molecular glue interface of AMP and 6-meTIMP showing cryo-EM density for the nucleotides. Right – rearrangement of PPAT interface residues in the 6-meTIMP structure (dark sidechains) compared to the AMP-bounds structure (light sidechains) e. Hydrophobic pocket of PPAT engaged by 6-meTIMP. f. 2D-ligand diagram of the 6-meTIMP molecular glue interface. g. PPAT activity assay measuring nucleotide-dependent inhibition in the presence of NUDT5 with 0.25 mM PRPP. Data points are the mean and error bars are SEM from n=3 independent experiments. h. Left – Western blot of endogenous NUDT5 3xFLAG immunoprecipitations following 16-hour treatment <t>with</t> <t>methotrexate</t> (2 µM), 6-MP (50 µM), and <t>MTX</t> + 6-MP. Right – Quantification of PPAT immunoprecipitation normalized to NUDT5 3xFLAG bait and compared to a DMSO-treated control condition. Data are individual values from n=3 independent biological replicate experiments and error bars are SEM. i. Fractional enrichment of AMP (M+2) and GMP (M+3) isotopologs in [ 15 N-amide]-glutamine labeling experiments conducted in the presence of 6-MP. Data points are individual values of n=6 biological replicates from two independent experiments and error bars are SEM. Statistical comparisons were performed using Welch’s two-tailed t-test with Bonferroni correction between wildtype and each mutant. *** denotes a Bonferroni adjusted p-value < 0.001 and ** is p-value < 0.01. j. FACS-based growth competition experiment comparing growth of ΔNUDT5 and endogenous L217A/K218A (LKAA) NUDT5 mutants to wildtype HEK293T treated with 6-MP and 6-TG. Data show n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments.
Methotrexate, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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methotrexate mtx  (Jiangsu Hengrui Medicine)
86
Jiangsu Hengrui Medicine methotrexate mtx
a. Simplified metabolism of 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). Asterisk denotes ability of 6-TGMP to be transformed into 6-meTGMP that may inhibit de novo purine synthesis. b. FACS-based growth competition comparing ΔNUDT5 and mutants to wildtype HEK293T cells treated with 6-TG. Data are individual values from n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments. c. Chemical structures of adenosine-5’-monophosphate (AMP) and 6-methylthioinosine-5’-monophosphate (6-meTIMP). d. Left – alignment of molecular glue interface of AMP and 6-meTIMP showing cryo-EM density for the nucleotides. Right – rearrangement of PPAT interface residues in the 6-meTIMP structure (dark sidechains) compared to the AMP-bounds structure (light sidechains) e. Hydrophobic pocket of PPAT engaged by 6-meTIMP. f. 2D-ligand diagram of the 6-meTIMP molecular glue interface. g. PPAT activity assay measuring nucleotide-dependent inhibition in the presence of NUDT5 with 0.25 mM PRPP. Data points are the mean and error bars are SEM from n=3 independent experiments. h. Left – Western blot of endogenous NUDT5 3xFLAG immunoprecipitations following 16-hour treatment <t>with</t> <t>methotrexate</t> (2 µM), 6-MP (50 µM), and <t>MTX</t> + 6-MP. Right – Quantification of PPAT immunoprecipitation normalized to NUDT5 3xFLAG bait and compared to a DMSO-treated control condition. Data are individual values from n=3 independent biological replicate experiments and error bars are SEM. i. Fractional enrichment of AMP (M+2) and GMP (M+3) isotopologs in [ 15 N-amide]-glutamine labeling experiments conducted in the presence of 6-MP. Data points are individual values of n=6 biological replicates from two independent experiments and error bars are SEM. Statistical comparisons were performed using Welch’s two-tailed t-test with Bonferroni correction between wildtype and each mutant. *** denotes a Bonferroni adjusted p-value < 0.001 and ** is p-value < 0.01. j. FACS-based growth competition experiment comparing growth of ΔNUDT5 and endogenous L217A/K218A (LKAA) NUDT5 mutants to wildtype HEK293T treated with 6-MP and 6-TG. Data show n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments.
Methotrexate Mtx, supplied by Jiangsu Hengrui Medicine, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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methotrexate mtx  (Shanghai Xinyi Bailuda Pharmaceutical Co Ltd)
86
Shanghai Xinyi Bailuda Pharmaceutical Co Ltd methotrexate mtx
a. Simplified metabolism of 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). Asterisk denotes ability of 6-TGMP to be transformed into 6-meTGMP that may inhibit de novo purine synthesis. b. FACS-based growth competition comparing ΔNUDT5 and mutants to wildtype HEK293T cells treated with 6-TG. Data are individual values from n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments. c. Chemical structures of adenosine-5’-monophosphate (AMP) and 6-methylthioinosine-5’-monophosphate (6-meTIMP). d. Left – alignment of molecular glue interface of AMP and 6-meTIMP showing cryo-EM density for the nucleotides. Right – rearrangement of PPAT interface residues in the 6-meTIMP structure (dark sidechains) compared to the AMP-bounds structure (light sidechains) e. Hydrophobic pocket of PPAT engaged by 6-meTIMP. f. 2D-ligand diagram of the 6-meTIMP molecular glue interface. g. PPAT activity assay measuring nucleotide-dependent inhibition in the presence of NUDT5 with 0.25 mM PRPP. Data points are the mean and error bars are SEM from n=3 independent experiments. h. Left – Western blot of endogenous NUDT5 3xFLAG immunoprecipitations following 16-hour treatment <t>with</t> <t>methotrexate</t> (2 µM), 6-MP (50 µM), and <t>MTX</t> + 6-MP. Right – Quantification of PPAT immunoprecipitation normalized to NUDT5 3xFLAG bait and compared to a DMSO-treated control condition. Data are individual values from n=3 independent biological replicate experiments and error bars are SEM. i. Fractional enrichment of AMP (M+2) and GMP (M+3) isotopologs in [ 15 N-amide]-glutamine labeling experiments conducted in the presence of 6-MP. Data points are individual values of n=6 biological replicates from two independent experiments and error bars are SEM. Statistical comparisons were performed using Welch’s two-tailed t-test with Bonferroni correction between wildtype and each mutant. *** denotes a Bonferroni adjusted p-value < 0.001 and ** is p-value < 0.01. j. FACS-based growth competition experiment comparing growth of ΔNUDT5 and endogenous L217A/K218A (LKAA) NUDT5 mutants to wildtype HEK293T treated with 6-MP and 6-TG. Data show n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments.
Methotrexate Mtx, supplied by Shanghai Xinyi Bailuda Pharmaceutical Co Ltd, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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methotrexate mtx  (Shanghai Macklin Biochemical)
86
Shanghai Macklin Biochemical methotrexate mtx
a. Simplified metabolism of 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). Asterisk denotes ability of 6-TGMP to be transformed into 6-meTGMP that may inhibit de novo purine synthesis. b. FACS-based growth competition comparing ΔNUDT5 and mutants to wildtype HEK293T cells treated with 6-TG. Data are individual values from n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments. c. Chemical structures of adenosine-5’-monophosphate (AMP) and 6-methylthioinosine-5’-monophosphate (6-meTIMP). d. Left – alignment of molecular glue interface of AMP and 6-meTIMP showing cryo-EM density for the nucleotides. Right – rearrangement of PPAT interface residues in the 6-meTIMP structure (dark sidechains) compared to the AMP-bounds structure (light sidechains) e. Hydrophobic pocket of PPAT engaged by 6-meTIMP. f. 2D-ligand diagram of the 6-meTIMP molecular glue interface. g. PPAT activity assay measuring nucleotide-dependent inhibition in the presence of NUDT5 with 0.25 mM PRPP. Data points are the mean and error bars are SEM from n=3 independent experiments. h. Left – Western blot of endogenous NUDT5 3xFLAG immunoprecipitations following 16-hour treatment <t>with</t> <t>methotrexate</t> (2 µM), 6-MP (50 µM), and <t>MTX</t> + 6-MP. Right – Quantification of PPAT immunoprecipitation normalized to NUDT5 3xFLAG bait and compared to a DMSO-treated control condition. Data are individual values from n=3 independent biological replicate experiments and error bars are SEM. i. Fractional enrichment of AMP (M+2) and GMP (M+3) isotopologs in [ 15 N-amide]-glutamine labeling experiments conducted in the presence of 6-MP. Data points are individual values of n=6 biological replicates from two independent experiments and error bars are SEM. Statistical comparisons were performed using Welch’s two-tailed t-test with Bonferroni correction between wildtype and each mutant. *** denotes a Bonferroni adjusted p-value < 0.001 and ** is p-value < 0.01. j. FACS-based growth competition experiment comparing growth of ΔNUDT5 and endogenous L217A/K218A (LKAA) NUDT5 mutants to wildtype HEK293T treated with 6-MP and 6-TG. Data show n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments.
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hypoxanthine methotrexate thymidine  (Thermo Fisher)
96
Thermo Fisher hypoxanthine methotrexate thymidine
a. Simplified metabolism of 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). Asterisk denotes ability of 6-TGMP to be transformed into 6-meTGMP that may inhibit de novo purine synthesis. b. FACS-based growth competition comparing ΔNUDT5 and mutants to wildtype HEK293T cells treated with 6-TG. Data are individual values from n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments. c. Chemical structures of adenosine-5’-monophosphate (AMP) and 6-methylthioinosine-5’-monophosphate (6-meTIMP). d. Left – alignment of molecular glue interface of AMP and 6-meTIMP showing cryo-EM density for the nucleotides. Right – rearrangement of PPAT interface residues in the 6-meTIMP structure (dark sidechains) compared to the AMP-bounds structure (light sidechains) e. Hydrophobic pocket of PPAT engaged by 6-meTIMP. f. 2D-ligand diagram of the 6-meTIMP molecular glue interface. g. PPAT activity assay measuring nucleotide-dependent inhibition in the presence of NUDT5 with 0.25 mM PRPP. Data points are the mean and error bars are SEM from n=3 independent experiments. h. Left – Western blot of endogenous NUDT5 3xFLAG immunoprecipitations following 16-hour treatment <t>with</t> <t>methotrexate</t> (2 µM), 6-MP (50 µM), and <t>MTX</t> + 6-MP. Right – Quantification of PPAT immunoprecipitation normalized to NUDT5 3xFLAG bait and compared to a DMSO-treated control condition. Data are individual values from n=3 independent biological replicate experiments and error bars are SEM. i. Fractional enrichment of AMP (M+2) and GMP (M+3) isotopologs in [ 15 N-amide]-glutamine labeling experiments conducted in the presence of 6-MP. Data points are individual values of n=6 biological replicates from two independent experiments and error bars are SEM. Statistical comparisons were performed using Welch’s two-tailed t-test with Bonferroni correction between wildtype and each mutant. *** denotes a Bonferroni adjusted p-value < 0.001 and ** is p-value < 0.01. j. FACS-based growth competition experiment comparing growth of ΔNUDT5 and endogenous L217A/K218A (LKAA) NUDT5 mutants to wildtype HEK293T treated with 6-MP and 6-TG. Data show n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments.
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methotrexate  (Merck & Co)
86
Merck & Co methotrexate
Protective capabilities of PRIOME ® JH and HT-PB01 in gut permeability using the C. elegans model. ( A ) Gut permeability protection effect represented by the percentage of fluorescence intensity of Nile red vs. control condition with damage. Gut damage was induced with <t>methotrexate</t> (MTX). Negative control (NC) was worms without damage and supplementation. Data are the mean ± SD of three independent experiments (N = a total of 90 worms per condition). Statistical analysis: Data were analyzed using a Kruskal–Wallis test, followed by Dunn’s multiple comparison test. The levels of significance were represented as: p -value < 0.0001 (****). ( B ) Representative images of Nile red staining in C. elegans under fluorescence microscopy. 20× magnification. Scale bar = 1000 µm.
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a. Simplified metabolism of 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). Asterisk denotes ability of 6-TGMP to be transformed into 6-meTGMP that may inhibit de novo purine synthesis. b. FACS-based growth competition comparing ΔNUDT5 and mutants to wildtype HEK293T cells treated with 6-TG. Data are individual values from n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments. c. Chemical structures of adenosine-5’-monophosphate (AMP) and 6-methylthioinosine-5’-monophosphate (6-meTIMP). d. Left – alignment of molecular glue interface of AMP and 6-meTIMP showing cryo-EM density for the nucleotides. Right – rearrangement of PPAT interface residues in the 6-meTIMP structure (dark sidechains) compared to the AMP-bounds structure (light sidechains) e. Hydrophobic pocket of PPAT engaged by 6-meTIMP. f. 2D-ligand diagram of the 6-meTIMP molecular glue interface. g. PPAT activity assay measuring nucleotide-dependent inhibition in the presence of NUDT5 with 0.25 mM PRPP. Data points are the mean and error bars are SEM from n=3 independent experiments. h. Left – Western blot of endogenous NUDT5 3xFLAG immunoprecipitations following 16-hour treatment with methotrexate (2 µM), 6-MP (50 µM), and MTX + 6-MP. Right – Quantification of PPAT immunoprecipitation normalized to NUDT5 3xFLAG bait and compared to a DMSO-treated control condition. Data are individual values from n=3 independent biological replicate experiments and error bars are SEM. i. Fractional enrichment of AMP (M+2) and GMP (M+3) isotopologs in [ 15 N-amide]-glutamine labeling experiments conducted in the presence of 6-MP. Data points are individual values of n=6 biological replicates from two independent experiments and error bars are SEM. Statistical comparisons were performed using Welch’s two-tailed t-test with Bonferroni correction between wildtype and each mutant. *** denotes a Bonferroni adjusted p-value < 0.001 and ** is p-value < 0.01. j. FACS-based growth competition experiment comparing growth of ΔNUDT5 and endogenous L217A/K218A (LKAA) NUDT5 mutants to wildtype HEK293T treated with 6-MP and 6-TG. Data show n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments.

Journal: bioRxiv

Article Title: Metabolic glues as a means of purine sensing and chemotherapeutic response

doi: 10.64898/2026.05.05.723063

Figure Lengend Snippet: a. Simplified metabolism of 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). Asterisk denotes ability of 6-TGMP to be transformed into 6-meTGMP that may inhibit de novo purine synthesis. b. FACS-based growth competition comparing ΔNUDT5 and mutants to wildtype HEK293T cells treated with 6-TG. Data are individual values from n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments. c. Chemical structures of adenosine-5’-monophosphate (AMP) and 6-methylthioinosine-5’-monophosphate (6-meTIMP). d. Left – alignment of molecular glue interface of AMP and 6-meTIMP showing cryo-EM density for the nucleotides. Right – rearrangement of PPAT interface residues in the 6-meTIMP structure (dark sidechains) compared to the AMP-bounds structure (light sidechains) e. Hydrophobic pocket of PPAT engaged by 6-meTIMP. f. 2D-ligand diagram of the 6-meTIMP molecular glue interface. g. PPAT activity assay measuring nucleotide-dependent inhibition in the presence of NUDT5 with 0.25 mM PRPP. Data points are the mean and error bars are SEM from n=3 independent experiments. h. Left – Western blot of endogenous NUDT5 3xFLAG immunoprecipitations following 16-hour treatment with methotrexate (2 µM), 6-MP (50 µM), and MTX + 6-MP. Right – Quantification of PPAT immunoprecipitation normalized to NUDT5 3xFLAG bait and compared to a DMSO-treated control condition. Data are individual values from n=3 independent biological replicate experiments and error bars are SEM. i. Fractional enrichment of AMP (M+2) and GMP (M+3) isotopologs in [ 15 N-amide]-glutamine labeling experiments conducted in the presence of 6-MP. Data points are individual values of n=6 biological replicates from two independent experiments and error bars are SEM. Statistical comparisons were performed using Welch’s two-tailed t-test with Bonferroni correction between wildtype and each mutant. *** denotes a Bonferroni adjusted p-value < 0.001 and ** is p-value < 0.01. j. FACS-based growth competition experiment comparing growth of ΔNUDT5 and endogenous L217A/K218A (LKAA) NUDT5 mutants to wildtype HEK293T treated with 6-MP and 6-TG. Data show n=3 biological replicates from a representative experiment. Similar results were obtained in two independent experiments.

Article Snippet: The following drugs and chemicals were used in this study at amounts specified in figures and legends: Pevonedistat; MLN4924 (MedChemExpress, HY-70062), methotrexate; MTX (MedChemExpress, HY-14519), lometrexol; LMX (MedChemExpress, HY-14521), brequinar (MedChemExpress, HY-108325) , rapamycin (Adooq Biosciences, A10782), 5-Phospho-D-ribose 1-diphosphate; PRPP (Sigma-Aldrich, P8296) , L-glutamine (Sigma-Aldrich, G8540); adenosine-5’-monophosphate; AMP (Sigma-Aldrich 01930), inosine-5’-monophosphate; IMP (MedChemExpress, HY-W010759), guanosine-5’-monophosphate; GMP (Sigma-Aldrich, G8377), AICA-ribonucleotide (Cayman Chemicals 33907), adenine (Thermo Scientific, A17622.14), hypoxanthine (MedChemExpress, HY-N0091), 6-thioguanine; 6-TG (Thermo Scientific, B21280.03), 6-mercaptopurine; 6-MP (Adooq Biosciences, A15898), 6-thioinosine-5’-monophosphate; 6-TIMP (Jena Biosciences, NU-1148), 6-methylthioinosine-5’-monophosphate; 6-meTIMP (Jena Biosciences, NU-1226), 6-methylthioguanosine-5’-monophosphate; 6-meTGMP (Jena Biosciences, NU-1128), 6-benzylthioinosine-5’-monophosphate; 6-benzylTIMP (WuXi, custom synthesis), 6-ethylthioinosine-5’-monophosphate 6-etTIMP (WuXi, custom synthesis), 6-ethylmercaptopurine riboside; 6-EMPR (WuXi, custom synthesis).

Techniques: Transformation Assay, Cryo-EM Sample Prep, Activity Assay, Inhibition, Western Blot, Immunoprecipitation, Control, Labeling, Two Tailed Test, Mutagenesis

a. Example cryo-EM density of the PPAT-NUDT5 6-meTIMP molecular glue interface with model fit. b,c. PPAT activity assay measuring inhibitory effects of 6-meTIMP in the presence and absence of wildtype NUDT5 and indicated mutants and c. compared to AMP only. d. Left – Representative Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with indicated drugs for 16 hours: methotrexate (MTX; 2 µM), lometrexol (LMX; 10 µM), 6-mercaptopurine (6-MP; 50 µM), MLN4924 (1 µM), brequinar (2 µM), and rapamycin (1 µM). Right – quantification of PPAT immunoprecipitation relative to NUDT5 3xFLAG bait and normalized to a DMSO-treated control condition. Data are individual values from n=3 biological replicates from independent experiments and error bars are SEM. e. Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with MTX (2 µM) for the indicated amounts of time. Similar results were obtained in two independent experiments. f. Western blot of endogenous PPAT 3xFLAG immunoprecipitations following 16-hour treatment with MTX (2 µM), 6-MP (50 µM), and MTX + 6-MP g. Time-resolved microscopy (incucyte) growth assays of wildtype and mutant HEK293T cells treated with the indicated drugs. Data are the mean and error bars are SEM of n=6 biological replicates. h. Levels of intracellular 6-TIMP and 6-meTIMP metabolites following 16-hour treatment with 6-MP (20 µM). Data are individual values and error bars are SEM from n=3 biological replicates. i. PPAT activity assay measuring inhibitory effects of 6-meTGMP in the presence and absence of wildtype NUDT5 and indicated mutants. Activity data shown in panels b, c and i are the mean and error bars are SEM of n=3 independent experiments.

Journal: bioRxiv

Article Title: Metabolic glues as a means of purine sensing and chemotherapeutic response

doi: 10.64898/2026.05.05.723063

Figure Lengend Snippet: a. Example cryo-EM density of the PPAT-NUDT5 6-meTIMP molecular glue interface with model fit. b,c. PPAT activity assay measuring inhibitory effects of 6-meTIMP in the presence and absence of wildtype NUDT5 and indicated mutants and c. compared to AMP only. d. Left – Representative Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with indicated drugs for 16 hours: methotrexate (MTX; 2 µM), lometrexol (LMX; 10 µM), 6-mercaptopurine (6-MP; 50 µM), MLN4924 (1 µM), brequinar (2 µM), and rapamycin (1 µM). Right – quantification of PPAT immunoprecipitation relative to NUDT5 3xFLAG bait and normalized to a DMSO-treated control condition. Data are individual values from n=3 biological replicates from independent experiments and error bars are SEM. e. Western blot of immunoprecipitations from endogenous NUDT5 3xFLAG HEK293T cells treated with MTX (2 µM) for the indicated amounts of time. Similar results were obtained in two independent experiments. f. Western blot of endogenous PPAT 3xFLAG immunoprecipitations following 16-hour treatment with MTX (2 µM), 6-MP (50 µM), and MTX + 6-MP g. Time-resolved microscopy (incucyte) growth assays of wildtype and mutant HEK293T cells treated with the indicated drugs. Data are the mean and error bars are SEM of n=6 biological replicates. h. Levels of intracellular 6-TIMP and 6-meTIMP metabolites following 16-hour treatment with 6-MP (20 µM). Data are individual values and error bars are SEM from n=3 biological replicates. i. PPAT activity assay measuring inhibitory effects of 6-meTGMP in the presence and absence of wildtype NUDT5 and indicated mutants. Activity data shown in panels b, c and i are the mean and error bars are SEM of n=3 independent experiments.

Article Snippet: The following drugs and chemicals were used in this study at amounts specified in figures and legends: Pevonedistat; MLN4924 (MedChemExpress, HY-70062), methotrexate; MTX (MedChemExpress, HY-14519), lometrexol; LMX (MedChemExpress, HY-14521), brequinar (MedChemExpress, HY-108325) , rapamycin (Adooq Biosciences, A10782), 5-Phospho-D-ribose 1-diphosphate; PRPP (Sigma-Aldrich, P8296) , L-glutamine (Sigma-Aldrich, G8540); adenosine-5’-monophosphate; AMP (Sigma-Aldrich 01930), inosine-5’-monophosphate; IMP (MedChemExpress, HY-W010759), guanosine-5’-monophosphate; GMP (Sigma-Aldrich, G8377), AICA-ribonucleotide (Cayman Chemicals 33907), adenine (Thermo Scientific, A17622.14), hypoxanthine (MedChemExpress, HY-N0091), 6-thioguanine; 6-TG (Thermo Scientific, B21280.03), 6-mercaptopurine; 6-MP (Adooq Biosciences, A15898), 6-thioinosine-5’-monophosphate; 6-TIMP (Jena Biosciences, NU-1148), 6-methylthioinosine-5’-monophosphate; 6-meTIMP (Jena Biosciences, NU-1226), 6-methylthioguanosine-5’-monophosphate; 6-meTGMP (Jena Biosciences, NU-1128), 6-benzylthioinosine-5’-monophosphate; 6-benzylTIMP (WuXi, custom synthesis), 6-ethylthioinosine-5’-monophosphate 6-etTIMP (WuXi, custom synthesis), 6-ethylmercaptopurine riboside; 6-EMPR (WuXi, custom synthesis).

Techniques: Cryo-EM Sample Prep, Activity Assay, Western Blot, Immunoprecipitation, Control, Microscopy, Mutagenesis

Protective capabilities of PRIOME ® JH and HT-PB01 in gut permeability using the C. elegans model. ( A ) Gut permeability protection effect represented by the percentage of fluorescence intensity of Nile red vs. control condition with damage. Gut damage was induced with methotrexate (MTX). Negative control (NC) was worms without damage and supplementation. Data are the mean ± SD of three independent experiments (N = a total of 90 worms per condition). Statistical analysis: Data were analyzed using a Kruskal–Wallis test, followed by Dunn’s multiple comparison test. The levels of significance were represented as: p -value < 0.0001 (****). ( B ) Representative images of Nile red staining in C. elegans under fluorescence microscopy. 20× magnification. Scale bar = 1000 µm.

Journal: Cells

Article Title: Heat-Treated Lacticaseibacillus rhamnosus Strains Modulate Inflammatory and Metabolic Processes in In Vitro Systems Relevant to Canine Osteoarthritis

doi: 10.3390/cells15040336

Figure Lengend Snippet: Protective capabilities of PRIOME ® JH and HT-PB01 in gut permeability using the C. elegans model. ( A ) Gut permeability protection effect represented by the percentage of fluorescence intensity of Nile red vs. control condition with damage. Gut damage was induced with methotrexate (MTX). Negative control (NC) was worms without damage and supplementation. Data are the mean ± SD of three independent experiments (N = a total of 90 worms per condition). Statistical analysis: Data were analyzed using a Kruskal–Wallis test, followed by Dunn’s multiple comparison test. The levels of significance were represented as: p -value < 0.0001 (****). ( B ) Representative images of Nile red staining in C. elegans under fluorescence microscopy. 20× magnification. Scale bar = 1000 µm.

Article Snippet: To induce intestinal permeability, L4-nematodes were exposed to 0.5 μg/mL methotrexate (MTX; Merck; St Louis, MO, USA) for 24 h. A condition without intestinal cell damage was included (NGM control medium).

Techniques: Permeability, Fluorescence, Control, Negative Control, Comparison, Staining, Microscopy