Figure 2. Deletion of B3GNT5 leads to depletion of nLc4 and P₁.

(A) B3GNT5-editing strategy using two different sgRNAs (red) contain PAM sequence (green) deleting a genomic region up- and downstream of the transcription start site (+1) including a part of the open reading frame (ORF). In silico analysis revealed a deletion of 898 bp. (B) Representative genotyping PCR for characterization of single cell clones. PCR numbers indicate the use of different primer pairs listed in experimental procedures. (C) Semi-quantitative PCR with two different primer pairs (B3GNT5_1 and B3GNT5_2) on wildtype (pIGROV1) and two representative knockout clones. SDHA was used as a reference gene. (D) DNA sequencing results for wildtype (WT), and selected biallelically deleted knockout clones (KO_1 and KO_2). (E) Representative counter plots for presence of Gb3, GM1, nLc4 and P₁ before (wildtype B3GNT5) and after gene disruption of B3GNT5 in IGROV1 cells (∆B3GNT5). Percentage of GSL-positive cells (FITC) is shown in each plot. Negative control (n.c.). (F) Box and Whisker plots representing CRISPR Cas9-mediated changes in expression of individual GSLs (percentage of FITC-positive cells, ordinate) for ∆B3GNT5 and parental cells (abscissa). ***p < 0.001 was obtained by two-way ANOVA. Data are representative out of four independent experiments. (G,H) Confocal fluorescence images displaying presence and absence of nLc4 (green, G, 40x magnification) and P₁ (green, H) in B3GNT5 wildtype (B3GNT5) and knockout (∆B3GNT5) cells, respectively. Cells are counterstained with DAPI (blue). Data are represented as mean ± s.d.