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Figure 1(A). Map of mutant eGFP gene, integrated in HCT 116 cells. The wild-type and mutant sequence of the eGFP gene, where the point mutation is in the eGFP cassette, TAG, are shown above (base is in bold and underlined). This is the target for the 47-mer oligonucleotide, EGFP3S/47NT, directed to the non-transcribed strand of the eGFP gene. EGFP3S/47NT is a single-stranded oligonucleotide with three phosphorothioate modifications on each terminus. (B). Selenomethionine is not cytotoxic in HCT 116 cells. Cells were incubated with selenomethionine at concentrations of 0, 50, 200, 400, and 1000 μM, respectively. Viability was determined by an MTT-reduction assay; each data point represent mean of three (± S.D.), non-treated cells are normalized to 100% viable. (C). Exposure to selenomethionine does not induce DSBs in HCT 116 cells. Cells were grown with respective treatment for 24 hours prior to harvesting and prepared for pulsed-field gel electrophoresis (PFGE). The gel was run for 24 hrs and subsequently stained with ethidium bromide. Lane M, S. cerevisiae chromosomal DNA size marker presented in kilobases; Lane C, non-treated control HCT 116 genomic DNA; Selenmethionine-treated (100 μM, 200 μM, 400 μM) HCT 116 cells; Lane MMS, MMS (150 μM) -treated HCT 116 genomic DNA.
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Figure 2(A). Selenomethionine treatment suppresses gene repair. HCT 116 cells were treated with selenomethionine (50 μM, 100 μM, 200 μM) for 24 hours prior to electroporation of the oligonucleotide. The cells were analyzed by FACS 24 hours after electroporation. The data represents three (± S.D.) independent experiments. (B). Selenomethionine induces mild, reversible G2/M delay. HCT 116 cells were not treated or treated with selenomethionine (50 μM, 200 μM) for 24 hours and processed for analysis of cell cycle. The profiles were generated using ModFit LT software. For cells treated with selenomethionine for 24 hrs: no treatment (57.6 +/- 1.4%G1, 26.1 +/- 0.6%S,16.37 +/- 0.8% G2), 50 μM selenomethionine (50.7 +/- 1.6% G1,24.7 +/- 1.3%S,24.6 +/- 2.6% G2) 200 μM selenomethionine (43.8 +/- 2.27% G1,20.1 +/- 3.9% S,36.1 +/- 6.2 % G2). For cells treated with selenomethionine for 24 hrs, washed, and allowed to recover for 24 hrs: no treatment (55.2 +/- 3.3% G1,28.8+/- 1.8S,16.1 +/- 1.4%G2), 50 μM selenomethionine (50.8 +/- 2.9% G1, 29.8+/- 3.3% S,19.4+/- 0.4%G2) 200 μM selenomethionine (53.2 +/- 1.2% G1, 31.7+/- 1.9% S,15.1+/- 0.7 G2).
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Figure 3 Selenomethionine treatment does not result in great levels of expressed or activated p53. HCT 116 cells were not treated or treated with selenomethionine (200 μM) for 24 hours prior to protein isolation. Western blot analysis reveals that expression of wild-type p53 is unchanged in the presence of selenomethionine; activation of p53 by serine-15 phosphorylation does not occur in the presence of selenomethionine; p53 is phosphorylated at serine-15 when cells are treated with camptothecin (CPT; 30 nM) for 24 hours; it serves here as a positive control.
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Figure 4 (A). Suppression of gene repair by p53 overexpression is equivalent to selenomethionine treatment. Cells were incubated with or without 200 μM selenomethionine for 24 hrs. 5 μg of respective overexpression plasmid (empty plasmid (pcDNA), wild-type p53 (P72), or mutant p53 (R175H)) and 8 μM EGFP3S/47NT oligonucleotide was electroporated in HCT 116 cells; gene repair levels were evaluated by FACS after 24 hours. (B). Selenomethionine treatment enhances gene repair in DLD-1 (p53+/- mutant). DLD-1 cells were treated with selenomethionine (50 μM, 100 μM, 200 μM) for 24 hours prior to electroporation of the oligonucleotide. The cells were analyzed by FACS 24 hours after electroporation. The data represents three (± S.D.) independent experiments.
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