Figure 4

ABCB1 as the primary resistant gene. A) Insertion sites near ABCB1 genomic locus are enriched in resistant samples. Insertion sites of a prescreened library and resistant pools in Chr7:85000000–89500000 are plotted. Scale is drawn as per Mb. Dot surfaces indicate number of positive samples, and y-axis indicates normalized read numbers as a percentage of total signals. Read numbers are unfiltered. All annotated genes within this region are shown as arrows. A blow-up view indicates ABCB1 genomic arrangement with open reading frame shown as yellow boxes. Asterisk denotes exon 3 with the ATG start codon (chr7:87229506). Insertion sites confirmed by TOPO cloning and Sanger sequencing are drawn as triangles above the diagram with direction of arrows indicating orientation of the CMV and the splice donor. HP, MP, TP1-3 denote HeLa, MCF7, and T47D paclitaxel resistant clones respectively. Colors of dots indicate orientation of the CMV with forward orientation relative to ABCB1 as blue and reverse orientation as yellow. B) PB insertions activate ABCB1 expression. Error bars show standard deviation (n=3). Significances are indicated by p-values. “pre” denotes prescreened libraries; MP, HP, and TP1-3 denote clones shown above. C) Detection of the chimeric mRNA in clones with insertions in the ABCB1 intron. Top panel (PB/ABCB1-E3) shows 404bp chimeric PCR products with a transposon-specific primer and an ABCB1 exon 3 primer. A lower band at 300bp could be due to alternative splicing. Bottom panel (hPBGD) shows the 151bp PCR products using the primer pair amplifying the porphobilinogen deaminase (PBGD) housekeeping gene. Three native cell lines (H, HeLa; M, MCF7; T, T47D) and a HeLa clone with PB insertions but not in the ABCB1 gene (HN) were used as controls. The first lane (MK) indicates 100bp DNA ladder (New England Biolabs).