Fig. 2: Validation of the Kat2-TGA-Fluc reporter.
From: Translational error in mice increases with ageing in an organ-dependent manner
A Readthrough induction by geneticin in HEK293 cells (HEK WT), embryonic stem cells (ES), and murine embryonic fibroblasts (MEF) expressing the Kat2-TGA-Fluc reporter. Left graph: Fluc activity, middle graph: Kat2 activity, right graph: Fluc/Kat2 ratio. Data shown represent the average of three independent experiments using 4 clones each transfected with the corresponding reporter construct; Fluc activities (measured in relative luminescent units RLU) and Kat2 activities (measured in relative fluorescent units RFU) are shown as mean ± SEM. B Fold induction of readthrough activity by geneticin in HEK WT, ES, and MEF; for calculation of fold induction, the Fluc/Kat2 expression ration in untreated cells was set to 1. C, D Readthrough induction in transgenic mice expressing the Kat2-TGA-Fluc reporter following geneticin treatment (closed diamonds, n = 4; 2 females (closed circles), 2 males (closed squares)) in comparison to control untreated transgenic mice (open diamonds, n = 4; 2 females (open circles), 2 males (open squares)), ratio RLU/RFU is shown as mean ± SEM. P values were calculated using two-sided Student’s t-test with equal variances. Δ P = 0.079, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, n.s. P > 0.05. Exact P-values are: (A) HEK WT—Fluc 8.8 × 10−7, Kat2 3.8 × 10−1, Fluc/Kat2 4.8 × 10−4; ES cells—Fluc 1.2 × 10−3, Kat2 4.5 × 10−2, Fluc/Kat2 4.3 × 10−4; MEF cells—Fluc 1.4 × 10−5, Kat2 8.5 × 10−1, Fluc/Kat2 4.6 × 10−6; (C) 4.7 × 10−2 for 4 h, 7.9 × 10−2 for 6 h, 2.1 × 10−2 for 8 h.
