Fig. 2

Sixteen novel anticancer genes cause cell death in transformed cells but not in normal cells. a Diagram depicting the process undertaken for the isolation of 16 novel anticancer genes. Screening was performed in two steps: in the first step a subset of 377 cell death inducing genes (in HEK-293 T cells) was transfected into non-transformed CV-1 cells in 4 consecutive rounds of screening where genes causing cell death above the internal threshold set for each round were gradually eliminated, resulting in a set of 78 gene candidates. DNA was isolated using silica oxide purification. In the second step, the plasmids were purified using standard DNA-miniPrep and another 35 genes were eliminated in CV-1 cells. The resulting 43 genes were transfected into HEK293T cells and 16 genes were selected for their ability to cause cell death in HEK-293T cells but not in CV-1 cells. b, c Relative cell death (CPRG ratio) in CV-1 (b) and HEK-293T (c) cells upon experimental overexpression of 16 anticancer genes. d, e Cell death in cervical cancer HeLa (d) and breast cancer MCF-7 cells (e), upon experimental overexpression of 16 anticancer genes. Cells were stained with Dioc₆ (early apoptosis) and PI (late apoptosis) and subjected to flow cytometry for PI positive and/or Dioc₆ negative cells. In all cases, luciferase (luc) was used for negative control and CAS2, CAS8 and RIP were used as positive controls. Histograms represent the average ± standard deviation (SD) of 3 independent transfections. Statistical significance was calculated using two-tailed Student’s t-test (*p ≤ 0.05, **p ≤ 0.005). pt: processed transcript, pr: partial overlap, nov: novel transcript