Fig. 1

Generation of novel immortal hPrEC cell lines designated p16-KO and Cl-3. (A) Scheme of the CDKN2A gene locus, which encodes two unrelated proteins p14^ARF and p16^INK4A via two separate promoters and alternative splicing of Exons 1α and 1β. Note that exon 2 is common but read in alternative frames. p14^ARF and p16^INK4A trigger two distinct senescence pathways that converge to induce G1 arrest. Two sgRNAs target exon 1α and 1β, which encode portions of p16^INK4A and p14^ARF proteins, respectively, another sgRNA targets the shared exon 2. (B) Morphology of primary hPrEC in culture and cell lines p16-KO and Cl-3 obtained upon selection with antibiotics. Each cell line was derived from a single colony, but the cell lines have not been single cell cloned, as they do not tolerate growth at low density. Cl-1 and Cl-2 were described previously.