TMEM165 KO cells showed reduced levels of glycosphingolipids.

(A-B) Both a TMEM165 KO cell line (TM-KO-3) and a mutant cell line (TM-Mut-1, insertion of Cys-Tyr residues between Leu279 and Cys280) showed increased resistance to Stx1 (A) and Stx2 (B). (C) TM-KO-3 and TM-Mut-1 cells showed reduced sensitivities to ricin (10 ng/mL) compared with WT cells. (D-E) TM-KO-3 and TM-Mut-1 cells showed similar sensitivities to A-Dtx (D) (Student’s t test, p > 0.05), but reduced sensitivity to Ctx (E) compared with WT cells. (F) TM-KO-3 and TM-Mut-1 cells showed reduced binding of Stx1 and CtxB. Ectopic expression of TMEM165 (with an N-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 TM-KO-3 and TM-Mut-1 cells have lower levels of Gb3, LacCer, GlcCer, Cer, and gangliosides (GM2) compared with WT cells. Quantification data are provided in S4 Data. All error bars indicate mean ± SD, N = 3.