Figure 6.

Feasibility of the CoIFI approach using a stably transformed cell line: (a) Schematic structure of the plasmid used to obtain stable human cell lines. ori: high copy number pUC origin in E. coli. Luc: Luciferase gene for a reporter assay. Ap: ampicillin resistance gene. TFT: Triplex Forming Tag. SV40: SV40 early enhancer/promoter. hyg^r: hygromycin resistance gene. NF-κB: a binding site for NF-κB proteins. (b) Reporter assay based on Luciferase activity. The G69 cells are stimulated by exposure to 10 ng/ml of TNFα for 2 hr; luminescence is measured by a luminometer. The data, derived from three independent assays, are expressed as relative light unit for non-stimulated cells (−TNFα) and stimulated cells (+TNFα). (c) Experimental design: Inputs are prepared from ≈0.9 × 10⁶ cells equivalent of cell lines containing the TFT site (+TFT; clone G69) or not (−TFT; clone c2-1). Input samples are incubated with either sTFO (scrambled sequence) or tTFO (target sequence). Together, three experimental conditions designed as a, b, c are tested (see main text for additional explanations). (d) Plasmid detection via PCR. 2% of the elution products are used as a template to amplify a DNA segment in the luciferase gene. Lanes a–c correspond to the conditions outlined in Fig. 6c. The indicated amounts of control plasmid pAS104.2, were similarly amplified. Quantification of the bands by Image Lab software (Bio-Rad) produces the standard curve shown in Supplementary Fig. S7. This experiment was performed independently five times and led to similar results. (e) Histone H3 detection via WB. ≈83% of the elution products and input from ≈ 50 cells equivalent are loaded. Lanes a~c correspond to the conditions outlined in Fig. 6c. (f) p65 detection via WB. Inputs are prepared from ≈2.5 × 10⁷ cells equivalent of clone G69 cell line. Extracts are incubated with either sTFO or tTFO. Input lane is loaded ≈ 200 cells equivalent. Full-length blots are presented in Supplementary Fig. S8.