Fig 1: OGT stabilizes FOXC1 protein via O-GlcNAcylation in NB cells. (A) Western blot assay showing the FOXC1 levels in SH-SY5Y cells stably transfected with scramble shRNA (sh-Scb) or sh-OGT #1, and those treated with CHX (20 μg·ml−1) as indicated. (B and C) Western blot assay indicating the expression of OGT and FOXC1 in SH-SY5Y cells stably transfected with sh-Scb or sh-OGT #1 (B) or incubated with OSMI-1 (C, 2.0 μmol·l−1), and those treated with MG132 (10 μmol·l−1) for 4 h. (D) Co-IP and Western blot assays revealing the binding of OGT to FOXC1 and FOXC1 O-GlcNAcylation in SH-SY5Y cells treated with control (CTL) DMEM/F12 or EBSS for 6 h. (E) Co-IP and Western blot assays showing the O-GlcNAcylation of FOXC1 in SH-SY5Y cells stably transfected with empty vector (mock), OGT, sh-Scb, sh-OGT #1, or sh-OGT #2. (F) In vitro O-glcNAcylation assay indicating the O-glcNAcylation of MBP-tagged FOXC1 protein in the presence of UDP-GlcNAc, GST-tagged OGT, or OGA. (G and H) Western blot assay showing the FOXC1 levels in SK-N-BE(2) (G) and SH-SY5Y (H) cells treated with TMG (G, 10 μmol·l−1) or OSMI-1 (H, 2.0 μmol·l−1), and those incubated with CHX (20 μg·ml−1) as indicated. (I) Co-IP and Western blot assays revealing the O-GlcNAcylation of FOXC1 in SH-SY5Y cells stably transfected with mock, Flag-tagged FOXC1, and that containing S8A or T68A mutation. (J to L) Dual-luciferase (J), ChIP-qPCR (K, normalized to input, n = 3), real-time qRT-PCR (K, normalized to β-actin, n = 5), and Western blot (L) assays showing the FOXC1 activity, FOXC1 enrichment, and target gene (ASNS or GPT2) expression in SH-SY5Y cells stably transfected with mock, Flag-tagged FOXC1, and that containing S8A or T68A mutation. (M) Schematic illustration indicating the action modes of OGT-mediated O-GlcNAcylation (G) of FOXC1 in regulating target gene (ASNS or GPT2) expression. ANOVA compared the difference in (J). **P < 0.01. Data are shown as representative of 3 independent experiments in (A) to (L).