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. 2019 May 9;9:7122. doi: 10.1038/s41598-019-43575-y

Figure 4.

Figure 4

APOBEC3B induces C>T|G>A mutations in lentivirally introduced genomic DNA in myeloma cells. (a,b) 3D-PCR analysis of mCherry genes (a) and puromycin resistance genes (puroR) (b) derived from RPMI8226 genomic DNA at 3 or 21 days post transduction with each shRNA lentivirus. 3D-PCR analysis of mCherry genes derived from AMO1 genomic DNA at 3 days post transduction with each shRNA lentivirus is also indicated in (a). (c) Mutation matrices of hyperedited mCherry sequences derived from RPMI8226 genomic DNA at 3 (upper panel) and 21 (lower panel) days post transduction with each shRNA lentivirus. The sequence data were obtained by performing TA cloning and Sanger sequencing. The first column indicates the bases before mutation, and the first line indicates the bases after mutation. The highlighted boxes indicate significant C-to-T or G-to-A substitutions. The sense strand of the mCherry sequence was used as a reference. (d) Sequence logo created with WebLogo indicating the frequencies of nucleotides adjacent to C-to-T mutation sites. (e) Deep sequencing analysis of the 3D-PCR products of mCherry-T2A-puroR genes derived from RPMI8226 genomic DNA at 21 days post transduction with each shRNA lentivirus. The Y axis indicates the proportion of each substitution in the total coverage, and the X axis indicates its location in the amplified gene from the EF1α promoter to the puroR gene. (f) Schema of Sanger sequencing results of each of the 10 clones of mCherry 3D-PCR products at 3 days post transduction with each shRNA lentivirus. G-to-A substitutions are indicated in green, C-to-T substitutions in red, G-to-C substitutions in light blue, and A-to-T substitutions in orange. The black squares indicate single-base deletions, whereas the dotted lines represent large deletions. The pink, purple or yellow boxes indicate microhomology at the DNA double-strand breakpoint. (g) 3D-PCR of EGFP derived from RPMI8226 genomic DNA at 10 days post transduction with control shRNA lentivirus under continuous incubation with the APOBEC3 inhibitors myricetin (5 μM) and aurintricarboxylic acid (ATA, 1 μM). We used lentiviral shRNA against APOBEC3B as a positive control.