TM9SF2 KO cells showed reduced levels of glycosphingolipids.
(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.