Fig 1: Upregulation of 5′-TOP mRNAs in p53−/− MEFs. p53fl/fl MEFs were transduced with control AAV-LacZ or AAV-Cre to knockout p53. (a) Polysome profiling showing increased polysome peaks following knockout of p53 (Cre). (b) Immunoblot analysis showing protein abundance of 5′-TOP encoded proteins (TPT1, RPL22, RPL23A, EEF2 and PABPC1) and 5′-TOP regulators (eIF4G1, LARP1, eIF4E and mTORC1) following knockout of p53 (AAV-Cre). (c) Luciferase activity of a 5′-TOP reporter is increased in p53−/− MEFs. Luciferase activity was normalized to firefly luciferase transfection control and set relative to LacZ control. Mean ± SD, n = 3. (d) qPCR shows no increase in mRNA expression of the 5′-TOP reporter. Normalized to firefly luciferase transfection control. Mean ± SD, n = 3. (e) Quantitation of blots in panel (b) for TPT1, RPL22, RPL23A, EEF2 and PABPC1. Total protein was used for normalization. Mean ± SD, n = 3–7. (f) qPCR shows no change in mRNA expression of 5′-TOP mRNAs following knockout of p53. Mean ± SD, n = 6. (g) Quantitation of blot in (b) and qPCR for LARP1. Mean ± SD, n = 7(protein), 6(RNA). (h) Quantitation of blot in (b) and qPCR for eIF4G1. Mean ± SD, n = 6(protein), 6(RNA). (i) Quantitation of blot in (b) and for mTORC1 pathway. Mean ± SD, n = 4. *, **, *** p-value < 0.05, < 0.01, < 0.001; two-tailed t-test with Bonferoni correction.