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. 2018 Jan 10;7:e31936. doi: 10.7554/eLife.31936

Figure 5. The signal-connectors specifically silence survival gene expression and inhibit cell growth in the targeted cancer cells.

(A) Mechanisms of the signal-connectors designed to selectively kill cancer cells, which control cell survival in response to the presence of activated TERTp and ligand. (B) A schematic representation of the genetic AND gate. hTERT promoter and ligand (1000 µM theophylline) are the two inputs of the circuit. (C) Two different signal-connectors were designed to target the 5ʹ-UTRs of human c-Myc mRNA and BCL2 mRNA. (D and E) Quantitative western blot analysis of targeted protein expression in bladder cancer cells (T24 and 5637) and normal fibroblast cells. NC, negative control vector with two repeated elements not having targets in the human genome. Blank, cells that were not transfected with vector. (F) Cell growth was measured by CCK-8 assay at different time intervals. ANOVA was used for the comparison of cell growth curves. Reported values are mean ± SD from three independent experiments.

Figure 5.

Figure 5—figure supplement 1. Sequence and cleavage mechanism of the ribozymes.

Figure 5—figure supplement 1.

The designed signal-connector sequence is inserted between the two ribozyme sequences. The two cleavage sites are indicated (red small arrows).The sequence of signal-connector can be released from the primary RNA transcript due to action of the ribozymes and therefore be free of potentially interfering flanking sequences.
Figure 5—figure supplement 2. Representative images of western blot analysis of c-Myc/BCL2 protein expression in bladder cancer cells transfected with the signal-connector or the controls.

Figure 5—figure supplement 2.

Figure 5—figure supplement 3. Representative images of western blot analysis of c-Myc/BCL2 protein expression in Fibroblast transfected with the signal-connector or the controls.

Figure 5—figure supplement 3.