Fig 2: 5'-TOP expression is unaffected by ARF knockdown in p53-/- MEFs. (a) Immunoblot analysis showing no change in protein abundance of PABPC1, TPT1 and RPL22 following ARF knockdown in p53-/- MEFs. (b) Quantitation of blots in panel (a). Total protein was used for normalization. Mean ± SD, n = 3. (c) No change in luciferase activity of a 5'-TOP reporter following ARF knockdown in p53-/- MEFs. Luciferase activity was normalized to Renilla luciferase transfection control and set relative to shSCR. Mean ± SD, n = 3. (d) Polysome profiling showing no change in polysome peaks in p53-/- MEFs following knockdown of ARF.

Fig 3: DHX36 is mainly cytoplasmic and does not directly interact with ribosomes. a Quantification of transgenic FLAG/HA(FH)-DHX36 expression upon 16 h induction with tetracycline (Tet.). TUBA4A-normalized DHX36 quantities are indicated below. b Endogenous (Endo.), as well as transgenic FH-tagged DHX36 isoform 1 (Iso. 1) and 2 (Iso. 2) show mainly cytoplasmic localization in biochemical fractionation experiments from HEK293 cells. Cytoplasmic (C) and nuclear (N) fractions were probed with anti-HISTH2B (nuclear marker), anti-TUBA4A (cytoplasmic marker), and anti-CANX (endoplasmic reticulum marker) antibodies. Endogenous DHX36 and transgenic FH-DHX36 isoforms 1 and 2 were detected with anti-DHX36 antibody and anti-HA antibody, respectively. c DHX36 can be co-purified with polyadenylated RNA. The RBP FMR1 served as a positive, TUBA4A as a negative control, respectively. d UV absorbances at 254 nm of RNase A-treated (red) and untreated (blue) HEK293 cell extracts separated by sucrose gradient centrifugation are shown. Peaks of UV absorbance corresponding to 40S, 60S, 80S ribosomes, and polysomes are indicated. Western blots probed for DHX36, RPL22, and EIF4A1 are shown below. Source data are provided as a Source Data file

Fig 4: 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.