Inhibition of FVIII production by the introduction of the patient variant in HEK293 cells. HEK293 cells were transfected with the plasmid for the expression of BDD F8 cDNA containing mini-intron 14 derived without or with the patient variant (c.5220-8563A>G). (A) Schematic presentation of F8 cDNA with mini-intron 14. The gray boxes indicate the abnormal exon. (B) Identification of aberrant F8 transcripts between exons 14 and 15 by RT-PCR in HEK293 cells with the variant. The red arrows indicate the size of the amplicons. (C) Relative expression of F8 mRNA between exons 25 and 26. Values represent the mean ± standard deviation (n = 3). Values represent the mean ± standard deviation (n = 3). Statistical analysis was performed by two-tailed Student t test. (D) FVIII expression within the cells was evaluated by immunohistochemical staining and photographed by conformal microscopy (Leica, TCS SP8). Red, FVIII; blue, 4′,6-diamidino-2-phenylindole (DAPI). The scales in merged images represent 10 μm. (E-G) FVIII activity (FVIII:C) (E), FVIII antigen (FVIII:Ag) (F), and the ratio of FVIII:C to FVIII:Ag (G) derived from the cell supernatant. Values are the mean ± standard deviation (n = 3). Statistical analysis was performed using two-tailed Student t test (comparison between 2 groups) or one-way analysis of variance with post hoc Tukey multiple comparison test (comparison between 2 values among 3 groups). (H) Immunoblotting of FVIII protein in the supernatant. The red and blue arrows represent the heavy chain (87.5 kilodalton [kDa]) and the light chain (79 kDa), respectively. Variant, HEK293 cells expressing F8 with the patient’s intronic variant; wild-type, HEK293 cells expressing F8 without the variant; control, parental HEK293 cells.