Extended Data Fig. 9: pUGDH(Y473) is required for tumour cell migration but not for proliferation.

a, UGDH-depleted A549 or H1299 cells were rescued with rUGDH(WT) or rUGDH(Y473F). b–d, UGDH-depleted H1299 cells rescued with rUGDH(WT) or rUGDH(Y473F) 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 SNAI1 mRNA. b, Relative precipitated RNA levels were normalized to those of the cells rescued with rUGDH(WT) without EGF treatment. c, d, Cells were treated with or without EGF (100 ng ml⁻¹) for the indicated times in the presence of actinomycin D (1 μg ml⁻¹). c, The remaining SNAI1 mRNA levels were examined by real-time PCR analyses. d, Transwell migration assays were performed with cells treated with or without EGF (100 ng ml⁻¹) for 24 h. e, UGDH-depleted A549 cells were rescued with rUGDH(WT) or rUGDH(Y473E) (left). Transwell migration assays were performed with cells treated with or without EGF (100 ng ml⁻¹) for 16 h (right). f, UGDH-depleted A549 or H1299 cells were rescued with rUGDH(WT) or rUGDH(Y473F). The cells rescued with rUGDH(Y473F) were infected with or without the lentivirus expressing SNAIL (left). Transwell migration assays were performed with cells treated with EGF (100 ng ml⁻¹) for 16 h (A549 cells) or 24 h (H1299 cells) (right). g, UGDH-depleted A549 cells rescued with rUGDH(WT) or rUGDH(Y473F) were stably infected with the lentivirus expressing luciferase. Subsequently, luciferase activities were determined. h, Luciferase-expressing, UGDH-depleted A549 cells rescued with rUGDH(WT) or rUGDH(Y473F) were implanted into randomized athymic nude mice by tail-vein injection (six mice per group). Representative images of H&E-stained sections in dissected lungs 35 days after inoculation are shown (top). The metastatic nodules were quantified based on the H&E-stained lung sections (bottom). Data represent the mean ± s.d. of the metastatic nodules per mouse in six mice (two-tailed Student’s t-test). i–k, UGDH-depleted A549 cells rescued with rUGDH(WT) or rUGDH(Y473F) were stably infected with the lentivirus expressing luciferase and then orthotopically implanted into the lungs of randomized athymic nude mice (six mice per group). i, Left, 15 days after inoculation, bioluminescence imaging of implanted mice was carried out and representative images of lung tumours are shown. Right, the luciferase intensities of lung tumours were statistically analysed. The relative luciferase intensities were normalized to those of the mice implanted with UGDH-depleted A549 cells rescued with rUGDH(WT). Data represent the mean ± s.d. of the luciferase intensities in six mice. j, Top, representative images of dissected lungs and H&E-stained sections in dissected lungs 15 days after inoculation are shown. Bottom, the percentage of tumour area was quantified based on the H&E-stained lung sections. The relative percentage of metastatic tumour area was normalized to that of mice implanted with cells rescued with rUGDH(WT). Data represent the mean ± s.d. of the relative percentage of tumour area per mouse in six mice. k, Left, representative images of IHC staining of tumours with anti-Ki-67 antibody are shown. Right, the relative percentages of Ki-67⁺ cells were quantified. Data represent the mean ± s.d. of the relative percentage of Ki-67⁺ cells in six mice (two-tailed Student’s t-test). a, e, f, Immunoblotting experiments were performed with the indicated antibodies. Data are representative of at least three independent experiments. b–g, Data represent the mean ± s.d. of three biologically independent experiments (two-tailed Student’s t-test).

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