Fig 2: miR-1236-3p negatively regulates TPT1 expression in cultured neurons. (A) The levels of miR-1236-3p expression in cultured neurons transfected with either miR-1236-3p inhibitor or inhibitor control. (B) TPT1 mRNA and (C) protein expression in cultured neurons transfected with either TPT1-siRNA or control-siRNA. (D) TPT1 mRNA and (E) protein expression in cultured neurons co-transfected with either miR-1236-3p inhibitor or inhibitor control and control-siRNA or TPT1-siRNA. **P<0.01 vs. inhibitor control; ##P<0.01 vs. Inhibitor + control-siRNA. miR, microRNA; TPT1, translationally-controlled tumor protein 1; siRNA, small interfering RNA.

Fig 3: 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 4: Effect of miR-1236-3p inhibitor and/or TPT1 knockdown on cell viability and apoptosis of cultured neurons in vitro. (A) Cultured neuronal cell viability was determined after 48 h using MTT assay. (B and C) Flow cytometry was used to analyze cultured neuronal cell apoptosis. (B) Representative dot plots in cultured neuronal cells co-transfected with either miR-1236-3p inhibitor or inhibitor control and TPT1-siRNA or control siRNA. (C) Quantified results of (B). **P<0.01 vs. inhibitor control; ##P<0.01 vs. Inhibitor + control-siRNA. miR, microRNA; TPT1, translationally-controlled tumor protein 1; siRNA, small interfering RNA; PI, propidium iodide; FITC, fluorescein isothiocyanate.

Fig 5: Association between miR-1236-3p and TPT1. (A) TargetScan software predicted a miR-1236-3p binding site in the 3′-UTR of TPT1. (B) A Dual-luciferase reporter assay was performed to validate the potential association between miR-1236-3p and TPT1 as proposed in (A), in neuronal cells co-transfected with either miR-1236-3p mimic or mimic control and WT TPT1 or MUT-TPT1. **P<0.01 vs. WT-TPT1 mimic control. (C) Western blot analysis of TPT1 protein expression. (D) Reverse transcription-quantitative PCR analysis of TPT1 mRNA expression. Data were presented as the mean ± standard deviation. **P<0.01 vs. Control. 3′-UTR, 3′-untranslated region; miR, microRNA; TPT1, translationally-controlled tumor protein 1; UTR, untranslated region; WT, wild-type; MUT, mutant-type.