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. 2018 Jun 21;37(43):5780–5793. doi: 10.1038/s41388-018-0375-0

Fig. 3.

Fig. 3

C1GALT1 regulates phosphorylation, EGF-binding affinity, and O-glycosylation of EGFR in HNSCC cells. a Effects of C1GALT1 on phospho-RTKs. SAS cells were transfected with non-targeting small interfering RNA (siRNA) (siCtr) or siRNA against C1GLAT1 (siC1GALT1-2) for 48 h and then treated with 10% FBS for 5 min after 12-h starvation. Upper panel, western blots showing C1GALT1 knockdown in SAS cells. Lower panel, phospho-RTK array. Decreased phospho-EGFR and phospho-MET were indicated. b Effects of C1GALT1 on EGF-induced phosphorylation of EGFR. Tyrosine phosphorylation of EGFR (pY1068) was analyzed in C1GALT1 overexpressing SAS cells, C1GALT1 knockdown OEC-M1 cells, and C1GALT1 knockout SAS cells. Cells were starved for 4 h and then treated with (+) or without (−) EGF (10 ng/mL) for 5 min. GAPDH was an internal control. c Scatchard plots of ligand-binding assays. SAS and OEC-M1 cells were transfected with non-targeting siRNA (siCtr) or siRNA against C1GLAT1 (siC1GALT1-2) and collected 72 h later for ligand-binding assays. The Kd was estimated by the binding data. d VVA pull-down assays of EGFR. Upper panel, changes in O-glycans on EGFR in C1GALT1 knockdown SAS and OEC-M1 cells. SAS and OEC-M1 cells were transfected with non-targeting siRNA or siRNAs against C1GALT1 (siC1GALT1-1 and siC1GALT1-2). Cell lysates were incubated with VVA-conjugated beads for 18 h and immunoblotted with an anti-EGFR antibody. Input EGFR, C1GALT1, and GAPDH were shown. Lower panel, changes in O-glycans on EGFR in C1GALT1 knockout SAS cells. Cell lysates were treated with or without neuraminidase, which was used to remove sialic acids. e Co-immunoprecipitation assays of C1GALT1 and EGFR. SAS and OEC-M1 cells were transfected with C1GALT1-HA/pcDNA3.1A for 48 h and collected. Lysates were immunoprecipitated (IP) with anti-HA or anti-EGFR antibody, as indicated, and then immunoblotted (IB) with anti-EGFR or anti-HA antibody. f SAS and OEC-M1 cells were transfected with empty pcDNA3.1 (Mock) or C1GLAT1/pcDNA3.1A (C1GALT1), treated with solvent control or 70 µM erlotinib, and then subjected to MTT assay. Cell viability at day 4 was shown. Data are analyzed by Student’s t-test. **P < 0.01. g SAS and OEC-M1 cells were transfected with empty pcDNA3.1 (Mock) or C1GLAT1/pcDNA3.1A (C1GALT1), treated with solvent control or 70 µM erlotinib, and then subjected to transwell migration, and Matrigel invasion assays. Data are analyzed by Student’s t-test. *P < 0.05. **P < 0.01