Figure 5.

Impaired HLA class I expression on a tapasin-deficient human colon cancer line. (A) Comparison between the tapasin-gene exon 2 sequencing traces of wild-type SW480 and its variant 3G12. The dash line starts with deletion/insertion mutation introduced by the CRISPR/Cas9-mediated genome editing system, followed by frameshift mutation occurred in both alleles. A solid line indicates the corresponding sequence of wild type showing homozygous alleles without mutation. (B) Western blot of SW480 and 3G12 treated with or without IFNγ, stained with tapasin- or actin-mAbs. (C) Flow cytometry of SW480 and 3G12 stained with an HLA-A24-specific c7709A2 mAb (pink), an HLA-A2-specific BB7.2 mAb (green), or without a primary antibody (black). D, Quantification of surface HLA class I levels. ΔMFI values on the y-axis were calculated from three independent experiments shown in Fig. 5D, by subtracting values of negative controls without a primary antibody. Data are shown as mean + SEM (n = 3). p-values were calculated using a two-tailed t-test (**p < 0.01). The mean-percentage changes of 3G12 compared with SW480 in ΔMFI of HLA-24 and HLA-A2 are also shown.