Extended Data Fig. 5: UGDH activity is required for HuR binding to SNAI1 mRNA.

a, A549 cells were transfected with SFB–SNAI1-ORF–3′ UTR(WT) or its deletion mutants, including SFB–SNAI1-ORF–3′ UTR(ΔARE1), SFB–SNAI1-ORF–3′ UTR(ΔARE2) or SFB–SNAI1-ORF–3′ UTR(ΔARE3). Cells were treated with EGF (100 ng ml⁻¹) for the indicated times in the presence of actinomycin D (1 μg ml⁻¹). The remaining SFB-SNAI1 mRNA levels were examined by real-time PCR analyses. b, Purified recombinant His–HuR (10 μg) was incubated with biotin–ARE1 (200 nM) for 4 h. Pull-down assays were performed with streptavidin agarose beads. c, H1299 cells with or without UGDH depletion were treated with or without EGF (100 ng ml⁻¹) for 30 min. Pull-down assays were performed by incubating the cell lysates with or without biotin–ARE1 for 4 h. d, A549 or H1299 cells with or without UGDH depletion were treated with or without EGF (100 ng ml⁻¹) for 4 h. RNA immunoprecipitation assays were performed in the cells using anti-HuR antibody, followed by real-time PCR analyses of precipitated SNAI1 mRNA. Relative precipitated RNA levels were normalized to those of the cells expressing shNT without EGF treatment. e, A549 cells were infected with the lentivirus expressing Flag–UGDH(WT) or Flag–UGDH(ED). Flag–UGDH(WT) or Flag–UGDH(ED) was immunoprecipitated using cell lysates for the measurement of enzymatic activity. f, g, UGDH-depleted A549 cells or H1299 cells were rescued with rUGDH(WT) or rUGDH(ED). f, The expression of UGDH was examined by immunoblotting analyses. g, The concentrations of intracellular UDP-Glc and UDP-GlcUA were determined. h, The concentrations of intracellular UDP-Glc and UDP-GlcUA were determined in A549 cells. i, UGDH-depleted H1299 cells were rescued with rUGDH(WT) or rUGDH(ED). The cells were treated with or without EGF (100 ng ml⁻¹) for 30 min. Pull-down assays were performed by incubating the cell lysates with or without biotin–ARE1 for 4 h. j, The complex of HuR with RNA (PDB code 4ED5) is superimposed on the HuR–UDP-Glc and HuR–UDP-GlcUA complexes. The protein and RNA are shown as cartoon and are coloured cyan and green, respectively. UDP-Glc and UDP-GlcUA are shown as sticks. UDP-Glc, UDP-GlcUA and RNA (U6–U10) in the two ligand binding sites are all represented by a van der Waals surface, and the van der Waals surfaces are coloured orange, pink and green, respectively. b, c, f, i, Immunoprecipitation and immunoblotting experiments were performed with the indicated antibodies. Data are representative of at least three independent experiments. a, d, e, g, h, Data represent the mean ± s.d. of three biologically independent experiments (two-tailed Student’s t-test).

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