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TM9SF2 KO cells showed reduced levels of glycosphingolipids.

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posted on 2018-11-27, 18:24 authored by Songhai Tian, Khaja Muneeruddin, Mei Yuk Choi, Liang Tao, Robiul H. Bhuiyan, Yuhsuke Ohmi, Keiko Furukawa, Koichi Furukawa, Sebastian Boland, Scott A. Shaffer, Rosalyn M. Adam, Min Dong

(A-B) TM9SF2 KO cells (SF2-KO-8 and SF2-KO-9) showed increased resistance to Stx1 (A) and Stx2 (B). SF2-WT-5 cells showed slightly reduced sensitivity to Stx1 and Stx2 compared with WT cells. Error bars indicate mean ± SD, N = 3. (C) SF2-KO-8 and SF2-KO-9 both showed lower sensitivities to ricin (10 ng/mL) compared with WT cells. (D-E) SF2-KO-8 and SF2-KO-9 cells showed similar sensitivities to A-Dtx (D) (Student’s t test, p > 0.05) but lower sensitivity to Ctx (E) compared with WT cells. (F) SF2-KO-8 and SF2-KO-9 cells showed reduced binding of Stx1 and CtxB. Ectopic expression of TM9SF2 (with a C-terminal triple HA tag) restored binding of Stx1 and CtxB in both cell lines. Scale bar, 5 μm. Representative images are from one of three independent experiments. (G) Mass spectrometry analysis revealed that SF2-KO-8 and SF2-KO-9 cells have lower levels of Gb3, LacCer, GlcCer, Cer, and GM2 compared with WT cells. Quantification data are listed in S4 Data. (H) SF2-KO-8 and SF2-KO-9 cells showed reduced levels of HS on cell surfaces, as measured by flow cytometry using an anti-HS antibody (10E4). LA-KO-10 and LA-KO-12 cells were also tested as controls. All error bars indicate mean ± SD, N = 3.

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