Extended Data Fig. 1: Characterization of B3GALT5-KO and B3GALT5-KO + OE cells.
From: Fucosylation of glycoproteins and glycolipids: opposing roles in cholera intoxication
(A, B) Representative histograms (left panel) from the flow cytometry analyses of cell surface binding of Lewis a antibody (A) or Lewis x antibody (B) to indicated cell lines. Bar graphs (right panel) show quantification from 3 independent trials. Error bars indicate mean ± SD. (C) Quantification of gMFI from flow cytometry analyses of cells treated with increasing concentrations of CTB. Data shown are from 3 independent trials and normalized to the maximum APC signal in WT cells. Error bars indicate mean ± SD. Indicated cell lines were incubated for 72 h with increasing concentrations of CTB-Saporin (D) or unconjugated saporin (E). Cell survival upon internalization of CTB-saporin was measured using the Cell Titer-Glo 2.0 assay. Data shown are luminescence values normalized to the signal from the untreated condition for each cell type. Each datapoint is a biological replicate consisting of 3 averaged technical replicates. Error bars indicate mean ± SD of 3 biological replicates. (F) Cells pretreated with BFA or vehicle control for 0.5 h were incubated for 1.5 h with CT (1 nM) or buffer alone. Accumulation of cAMP was measured. Data shown are inverse of luminescence values normalized first to the total amount of cells plated for each cell line, then to the signal in CT-treated control cells. Each datapoint is a biological replicate consisting of 3 averaged technical replicates. Error bars indicate mean ± SD of 3 biological replicates. (G) Control, B3GALT5-KO m1, m2 and KO + OE cells were treated with forskolin (10 µM) for 0.5 h and then analyzed as in panel F. Statistical analyses for panels A and B were performed by one-way ANOVA with Tukey correction and for panels C, F, and G by two-way ANOVA with Tukey correction.‘ns’ indicates not significant, **** indicates adjusted P-value < 0.0001. Exact P-values are as follows: 0.0004 and 0.0412 for control versus B3GALT5-KO m2 and B3GALT5-KO + OE; 0.0041 and 0.0024 for B3GALT5-KO m1 versus B3GALT5-KO m2 and B3GALT5-KO + OE, respectively (panel B); 0.0390 for control versus B3GALT5-KO m2 treated with 0.0375 μg/mL; 0.0002 and 0.0014 for control versus B3GALT5-KO m2 or m1 treated with 0.075 μg/mL, respectively (panel C).
