Figure 1. Evaluation of the CRISPR/Cas9-generated CaCo-2 LSR knockout cells.

(A) DNA sequencing chromatograms for CaCo-2_WT and CaCo-2_ΔLSR cells covering a sequence within exon 2 of the LSR gene. Grey dotted line indicates the Cas9 target sequence. A red box highlights the base pair insertion (thymine) in exon 2 of the LSR gene from CaCo-2_ΔLSR cells. (B) LSR immunoblot with whole-cell lysates from CaCo-2_WT (WT) and CaCo-2_ΔLSR (ΔLSR) cells (top panel). Equal loading of samples was verified by detecting tubulin with a specific antibody (bottom panel). (C) Intoxication of CaCo-2_WT and CaCo-2_ΔLSR cells with CDT. Microscopic images were taken at indicated time points after toxin addition. (D) Suspensions of CaCo-2_WT and CaCo-2_ΔLSR cells were incubated with the receptor-binding domain of CDT labeled with DyLight488 (RBD_DL488) or were left untreated (mock) prior to FACS analysis. FACS results are presented as histogram plots and single cell events (cells) are plotted against the intensity of cell-surface bound fluorescence (Log FL intensity). White peaks indicate the fluorescence distribution of cells incubated with RBD_DL488 and grey peaks represent mock-treated cells with no addition of fluorescence-labeled protein.