Extended Data Fig. 2: UGDH regulates SNAI1 mRNA stability.
From: UDP-glucose accelerates SNAI1 mRNA decay and impairs lung cancer metastasis
a, A549 or H1299 cells were stably infected with the lentivirus expressing Flag–SLC35D1. Representative images of immunofluorescence staining with anti-Flag and anti-GLUT1 antibodies from three independent experiments are shown. b–e, A549 or H1299 cells stably expressing shNT or shUGDH were stably infected with or without the lentivirus expressing SLC35D1. b, Protein expression of SLC35D1 and UGDH was examined. c–e, Cells were then supplemented with or without UDP-GlcUA (5 mM) for 16 h (A549) or 24 h (H1299). c, Intracellular UDP-GlcUA concentrations were determined. d, Cellular hyaluronic acid concentrations were determined. e, Transwell migration assays were performed in the H1299 cells treated with or without UDP-GlcUA for 24 h. f, A549 cells stably expressing shNT or shUGDH were supplemented with increasing concentrations of hyaluronic acid for 16 h. The concentrations of cellular hyaluronic acid were determined (left). Transwell migration assays were performed in the presence of increasing concentrations of hyaluronic acid for 16 h (right). g, The mRNA levels of the EMT-related genes CDH1, CDH2, SNAI1, SNAI2, TWIST1, ZEB1, ZEB2, VIM, TCF3, TCF4, FOXC2, SIX1, GRHL2 and ELF5 were examined in A549 cells with or without UGDH depletion by real-time PCR analyses. h, The mRNA levels of SNAI1, CDH1 and CDH2 were examined in UGDH-depleted H1299 cells rescued with or without rUGDH by real-time PCR analyses. i, Protein expression of SNAIL, N-cadherin and E-cadherin was examined in UGDH-depleted A549 or H1299 cells rescued with or without rUGDH by immunoblotting analyses. j, UGDH-depleted A549 cells rescued with or without rUGDH were transfected with a SNAI1 promoter–luciferase reporter construct. Luciferase activities were determined. Relative luciferase activities were normalized to the luciferase activities of tumour cells expressing shNT and the luciferase activities of the Renilla control plasmid. k, UGDH-depleted H1299 cells were rescued with or without rUGDH. The mRNA stability of SNAI1 was determined by treating cells with actinomycin D (1 μg ml−1). The remaining SNAI1 mRNA levels were examined at the indicated time points by real-time PCR analyses. b, i, Immunoblotting experiments were performed with the indicated antibodies. Data are representative of at least three independent experiments. c–h, j, k, Data represent the mean ± s.d. of three biologically independent experiments (two-tailed Student’s t-test).
