Extended Data Figure 2. ATTAC lacks promoter elements required for expression in replication competent cells or aged lymphocytes expressing high levels endogenous p16Ink4a.
(a–c) SV40 Large-T antigen-immortalized ATTAC MEFs robustly express endogenous p16Ink4a (due to SV40 Large T-mediated inactivation of Rb) but fail to engage the ATTAC transgene and are not subject to FKBP-Casp8-mediated elimination. a, p16Ink4a protein levels in p4 primary MEFs and MEFs immortalized with SV40 Large-T antigen. Actin was used as a loading control. b, p16Ink4a protein levels in immortalized MEFs treated with vehicle or two concentrations of AP. Actin was used as a loading control. c, Expression of ATTAC and senescence marker transcripts in p4 primary MEFs, vehicle-treated immortalized MEFs, and AP-treated immortalized MEFs (n = 3 independently generated MEF lines per group). d, Schematic representation of the endogenous p16Ink4a locus and the various p16Ink4a promoter regions driving ATTAC, 3MR and Firefly luciferase (FLUC). ATTAC and p16-3MR mice have 2.6 kb and ~50 kb p16Ink4a promoter fragments driving transgene activity, respectively. p16-FLUC has firefly lucif-erase knocked into the endogenous p16Ink4a locus, which keeps the entire promoter region intact but ablates p16Ink4a protein expression. e, p16Ink4a protein levels in early passage primary and SV40 Large-T antigen-immortalized p16-3MR MEFs. f, Expression of senescence marker mRNA in early and late passage primary MEFs and SV40 Large-T antigen-immortalized p16-3MR MEFs (n = 1 independently generated MEF line per group performed in triplicate). g, Expression of senescence marker mRNA in early and late passage primary MEFs and SV40 Large-T antigen-immortalized p16-FLUC MEFs (n = 1 independently generated MEF line per group performed in triplicate). h, Expression of ATTAC and senescence markers in CD3+ T-cells from 12 and 18-month-old ATTAC mice (n = 5 mice per group). Error bars indicate s.e.m. Statistical significance was determined by unpaired two-tailed t tests. *, P<0.05; **, P<0.01; ***, P<0.001. We note that all values in f and g have a P<0.05 compared to p4 MEFs, with the exception of the one marked NS for not significant. For gel source data, see Supplementary Fig. 1.