Extended Data Fig. 6: UDP-Glc inhibits mRNA binding of HuR and tumour metastasis.

a, Isothermal titration calorimetry assays were performed with His–RRM1/2(WT) (0.05 mM), His–RRM1/2(Y63F) (0.05 mM) or His–RRM1/2(N134A) (0.05 mM) and UDP-Glc (0.5 mM, left) or UDP-GlcUA (0.5 mM, right). Data are representative of three independent experiments. b, Purified recombinant His–RRM1/2(WT) or His–RRM1/2(N134A) (2 μg) was incubated with biotin–ARE1 (0.5 μM) for 4 h, followed by pull-down assays. c, Purified recombinant His–RRM1/2(WT) or His–RRM1/2(N134A) (2 μg) was incubated with biotin–ARE1 (5 μM) for 4 h in the absence or presence of UDP-Glc (25 μM), followed by pull-down assays. d, e, ELAVL1-depleted A549 or H1299 cells were rescued with rHuR(WT) or rHuR(N134A). d, The expression of HuR was examined. e, 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. f, ELAVL1-depleted H1299 cells rescued with rHuR(WT) or rHuR(N134A) were treated with or without EGF (100 ng ml⁻¹) for 4 h. RNA immunoprecipitation assays were performed using an anti-HuR antibody, followed by real-time PCR analyses of precipitated SNAI1 mRNA. Relative precipitated RNA levels were normalized to those of the cells rescued with rHuR(WT) without EGF treatment. g, ELAVL1-depleted A549 cells rescued with rHuR(WT) or rHuR(N134A) were treated with or without EGF (100 ng ml⁻¹) for 4 h. RNA immunoprecipitation assays were performed using anti-HuR antibody, followed by real-time PCR analyses of precipitated mRNA transcribed by indicated genes. Relative precipitated RNA levels were normalized to those of the cells rescued with rHuR(WT) without EGF treatment. h, i, ELAVL1-depleted H1299 cells rescued with rHuR(WT) or rHuR(N134A) were treated with or without EGF (100 ng ml⁻¹) for the indicated times in the presence of actinomycin D (1 μg ml⁻¹). h, The remaining SNAI1 mRNA levels were examined by real-time PCR analyses. i, Transwell migration assays were performed with cells treated with or without EGF (100 ng ml⁻¹) for 24 h. j, Cell proliferation of ELAVL1-depleted A549 cells rescued with rHuR(WT) or rHuR(N134A) was determined. k, ELAVL1-depleted A549 cells rescued with rHuR(WT) or rHuR(N134A) were stably infected with the lentivirus expressing luciferase. Subsequently, luciferase activities were determined. l, Luciferase-expressing, ELAVL1-depleted A549 cells rescued with rHuR(WT) or rHuR(N134A) were implanted into randomized athymic nude mice by tail-vein injection (six mice per group). Top, representative images of H&E-stained sections in dissected lungs 30 days after inoculation are shown. Bottom, the metastatic nodules were quantified based on the H&E-stained lung sections. Data represent the mean ± s.d. of the metastatic nodules per mouse in six mice (two-tailed Student’s t-test. b–e, Immunoblotting experiments were performed with the indicated antibodies. Data are representative of at least three independent experiments. f–k, Data represent the mean ± s.d. of three biologically independent experiments (two-tailed Student’s t-test).

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