Extended Data Fig. 10: pUGDH(Y473) correlates with SNAIL expression and metastatic recurrence.

a, UGDH-depleted A549 cells rescued with rUGDH(WT) or rUGDH(Y473F) (pGIPZ-shUGDH also contains the GFP ORF) were implanted into randomized athymic nude mice by tail-vein injection (five mice per group). Subsequently, 3 h after inoculation, the mice were euthanized. Representative images of intravascular tumour cells (green) out of lung blood vessels (red) are shown. The numbers of tumour cells in lungs (including intracellular and extravascular tumour cells) were quantified. Data represent the mean ± s.d. of the relative number of tumour cells per mouse in five mice normalized to the control group. b, c, Cell proliferation (b) and colony formation (c) were determined in A549 cells stably expressing shNT or shUGDH, or UGDH-depleted A549 cells rescued with rUGDH(WT) or rUGDH(Y473F). d, A549 cells stably expressing shNT or shUGDH, or UGDH-depleted A549 cells rescued with rUGDH(WT) or rUGDH(Y473F) were subcutaneously injected into randomized athymic nude mice (six mice per group). The weight of xenograft tumours was statistically analysed. Data represent the mean ± s.d. of relative tumour weight in six mice (two-tailed Student’s t-test). e, Validation of antibody specificities. IHC analyses of human lung cancer specimens were performed with the indicated antibodies in the presence or absence of specific blocking peptides or proteins. Data are representative of three biologically independent experiments. f, g, IHC analyses of 114 specimens from patients with lung cancer were performed using anti-pUGDH(Y473) and anti-SNAIL antibodies. f, Representative images of IHC staining of three lung cancer specimens are shown. g, Semi-quantitative scoring (using a scale from 0 to 300) was carried out, Pearson’s correlation test. h, IHC analyses were performed in primary tumours from patients with lung cancer with or without metastatic recurrence using anti-pUGDH(Y473) antibody. Staining scores of pUGDH(Y473) were compared between the patients with metastatic recurrence (n = 50) and the patients without metastatic recurrence (n = 50). Data represent the mean ± s.d. of the two groups (two-tailed Student’s t-test). i, IHC staining was performed in primary tumours and paired metastatic tumours from 16 patients with lung cancer with distant metastasis using an anti-pUGDH(Y473) antibody. Staining scores of the primary lung cancer specimens were compared with those of metastatic lung cancer specimens. Data represent the mean ± s.d. of the two groups (two-tailed paired Student’s t-test). j, Schematic model of the mechanism of UGDH-regulated tumour cell migration. Upon EGFR activation, phosphorylated UGDH(Y473) interacts with HuR and converts UDP-Glc into UDP-GlcUA, which releases HuR to bind to and stabilize SNAI1 mRNA. Increased SNAIL expression consequently promotes tumour cell migration. Exo, exonuclease. b, c, Data represent the mean ± s.d. of three biologically independent experiments (two-tailed Student’s t-test).

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