Impaired suppressive activities of human MUTYH variant proteins against oxidative mutagenesis

Figure 1 Establishment of H1299 human cell lines inducibly expressing MUTYH variant proteins. A: Detection of MUTYH proteins in cumate-inducible stable cell lines expressing MUTYH in the presence of cumate using Western blotting analysis with an anti-MUTYH antibody. Lysates from empty vector-transposed cells and cells inducibly expressing wild-type (WT) MUTYH or p.R154H, p.M255V, p.L360P, or p.P377L MUTYH variants were analyzed. β-tubulin protein was also analyzed as an internal control; B: Immunofluorescence detection of MUTYH proteins expressed in the cell lines used in (A) in the presence of cumate. The MUTYH protein (red) was stained with anti-MUTYH as the primary antibody and Alexa Fluor 594-conjugated goat anti-mouse IgG as the secondary antibody. The nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (blue); C: Immunofluorescence detection of endogenous MUTYH proteins in the empty vector-transposed cells as described in (B). The intensity of the signals of MUTYH protein (red) was enhanced with image-editing software to determine the subcellular localization of endogenous MUTYH protein. The nuclei were counterstained with DAPI (blue).

Figure 2 Nuclear localization of MUTYH variant proteins (p. R154H, p.M255V, p.L360P, and p.P377L). H1299 cells were transiently transfected with a vector expressing various types of MUTYH proteins tagged with FLAG, and MUTYH-FLAG protein (red) was stained with anti-FLAG M2 as the primary antibody and Alexa Fluor 594-conjugated goat anti-mouse IgG as the secondary antibody. The nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI) (blue). WT: Wild-type.

Figure 3 Measurement of the mutation frequency of the supF gene in the pMY189 plasmid using a supF forward mutation assay in H1299 human cell lines inducibly expressing MUTYH variant proteins. Empty vector-transposed cells and cells inducibly expressing wild-type (WT) MUTYH or p.R154H, p.M255V, p.L360P, or p.P377L MUTYH variants in the presence of cumate were transfected with a pMY189 shuttle plasmid, and the mutation frequency of supF in these human cell lines was measured. “8-hydroxyguanine (8OHG)” indicates a pMY189 plasmid containing an 8OHG residue at position 159 of supF, while “G” indicates a pMY189 plasmid containing the WT supF gene. The data are shown as the means ± SE.