Fig. 2

ACD p.G223V protects from camptothecin-induced apoptosis and increases telomere length. a. Schematic representation of the ACD protein. The p.G223V mutation, depicted in black, is adjacent to the TEL patch of the OB-fold domain involved in telomerase recruitment and composed of seven critical amino acids located in a region defined by the curly bracket (E168, E169, E171, R180, L183, L212 and E215) [30]. p.Q320X, p.P491T and p.K170del, depicted in grey, are three germline mutations recently identified and associated with familial melanomas and bone marrow failure disorders [34–36]. TPP1C corresponds to the TIN2-binding domain. Together, the OB (oligonucleotide/oligosaccharide-binding) and PBD (POT1 binding domain) domains form the ACDN domain necessary for POT1 binding to telomeric DNA and the stimulation of telomerase processivity. b. In vitro apoptosis assays show overall reduced levels of apoptosis associated with ACD p.G223V. The c.659 g > t mutation was introduced into the ACD transgene by site-directed mutagenesis and expressed in Nalm-6 cells. The graph shows annexin V/PI staining for 3 h on Nalm-6 pLenti (empty vector), Nalm-6 ACD WT and Nalm-6 ACD G223V cells. c. The telomere restriction fragment assay (TRF) showed a quantitative increase in telomere size for Nalm-6 ACD G223V cells at passage 16 (p16) and p27 after selection. Mean TRF length = ∑ (ODi)/∑ (ODi/Li) where ODi is the radioactive signal, Li is the TRF fragment length at position i. The bar chart of Fig. 2c (bottom) represents the mean TRF length for each condition directly quantified from each corresponding lane of the TRF gel presented at the top of Fig. 2c Significance (in b and c) was determined by a Mann–Whitney U test; p-values <0.05 are represented by an asterisk