Fig 1: ADSL is the critical gene targeted by miR-21 in regulating purine metabolism.A Computational prediction of RNA duplex formation between miR-21-3p and ADSL mRNA’s 3’UTR (untranslated region). Mutations generated within the 3’UTR for the luciferase assay are shown in red. WT, wild-type. B Dual-luciferase reporter assay in H1975 cells treated with the miR-21-3p inhibitor. The Firefly luciferase and Renilla luciferase activities were measured 48 h post-co-transfection with the locked nucleic acid (LNA) miR-21-3p inhibitor (anti-miR-21-3p) or a control plasmid (anti-miR-control) and WT or mutant ADSL 3’UTR. N = 3 replicates. C Dual-luciferase reporter assay in H1975 cells treated with the miR-21-5p inhibitor. The Firefly luciferase and Renilla luciferase activities were measured 48 h post-co-transfection with the locked nucleic acid (LNA) miR-21-5p inhibitor (anti-miR-21-5p) or a control plasmid (anti-miR-control) and wild-type (WT) or mutant ADSL 3’UTR. N = 5 replicates. D Western blot analysis and quantification of ADSL in H1975 parental and osimertinib-tolerant (OTR) cells. β-Actin was used as a loading control. N = 3 replicates. E Western blot analysis and quantification of ADSL in H1975 OTR cells treated with the LNA miR-21-5p inhibitor (anti-miR-21-5p) or a control plasmid (anti-miR-control). β-Actin was used as a loading control. N = 2 replicates. F Western blot analysis and quantification of ADSL in parental H1975 cells treated with the LNA inhibitor against miR-21-3p (anti-miR-21-3p), miR-21-5p (anti-miR-21-5p), and a miRNA control (anti-miR-control). β-Actin was used as a loading control. N = 2 replicates. G A correlation between MIR21 and ADSL gene expression in lung tumor tissues from patients. RNA expression data were extracted from three independent RNA transcriptomics datasets (GSE3141, GSE16534, and GSE8894) by the Lung Cancer Explorer web portal. Each dot represents one patient’s sample. H A diagram describing the purine de novo synthesis pathway and nucleotide cycle reactions. The enzymes in green and blue colors are predicted targets of miR-21-5p and miR-21-3p by the TargetScan analysis. The enzymes in gray color are non-predicted targets of either miR-21-5p or miR-21-3p. I Western blot analysis and quantification of PPAT, GART, PFAS, PAICS, ATIC, AMPD2, and ADSS in H1975 parental and OTR cells. β-Actin shown in (D) was used as the loading control. N = 2 replicates. J Western blot analysis and quantification of PPAT, GART, PFAS, PAICS, ATIC, AMPD2, and ADSS in H1975 OTR cells treated with the LNA miR-21-5p inhibitor (anti-miR-21-5p) or a control plasmid (anti-miR-control). β-Actin shown in (E) was used as the loading control. N = 2 replicates. K Western blot analysis and quantification of MTHFD1 and MTHFD2 in H1975 parental and OTR cells. β-Actin shown in (D) was used as the loading control. N = 2 replicates. L Western blot analysis and quantification of MTHFD1 and MTHFD2 in H1975 OTR cells treated with the LNA miR-21-5p inhibitor (anti-miR-21-5p) and a miRNA control plasmid (anti-miR-control). β-Actin shown in (E) was used as the loading control. N = 2 replicates. M Western blot analysis and quantification of PPAT, GART, and PAICS in H1975 parental cells treated with the LNA inhibitor against miR-21-3p (anti-miR-21-3p), miR-21-5p (anti-miR-21-5p), and a miRNA control (anti-miR-control). β-Actin shown in (F) was used as the loading control. N = 3 replicates. Data are mean ± s.e.m. and were analyzed with unpaired two-tailed t-test (B–E, I–L); Pearson correlation analysis (G); RM one-way ANOVA (F, M).
Supplier Page from Abcam for Anti-AdSS 2 antibody [EPR12331-52]