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. Author manuscript; available in PMC: 2013 Oct 25.
Published in final edited form as: Oncogene. 2012 Jun 18;32(17):2169–2178. doi: 10.1038/onc.2012.238

Figure 3. RNPC1 destabilizes the MDM2 transcript.

Figure 3

(A) The level of MDM2 transcript is decreased by RNPC1 in p53−/− HCT116 cells. Total RNAs were isolated from p53−/− HCT116 cells uninduced or induced to express RNPC1 for 48 h, followed by cDNA synthesis. Left panel: Semi-quantitative RT-PCR analysis was performed to determine the level of RNPC1, MDM2, and actin. Right panel: Quantitative RT-PCR analysis was performed in triplicates and the level of MDM2 transcript was normalized to that of GAPDH control. The relative fold change of MDM2 transcript was a ratio by dividing the level of MDM2 transcript in RNPC1-expressing cell to that in control cells. (B) Inducible knockdown of RNPC1 increases the level of MDM2 transcript in SW480 cells. Total RNAs were isolated from SW480 cells uninduced or induced to express RNPC1 shRNA for 3 days, followed by cDNA synthesis. Left panel: The level of RNPC1, MDM2, and actin transcripts was determined by semi-quantitative RT-PCR analysis. Right panel: Quantitative RT-PCR analysis was performed in triplicates and the level of MDM2 transcript was normalized to that of GAPDH control. The relative fold change of MDM2 transcript was a ratio by dividing the level of MDM2 transcript in RNPC1-knockdown cells to that in control cells. (C) The level of MDM2 transcript is increased in PC3 cells upon RNPC1 knockdown. The experiment was performed as in (B) except that PC3 cells were transiently transfected with scrambled or RNPC1 siRNA for 3 days. (D) RNPC1 deletion in primary MEFs increases the level of MDM2 transcript independent of p53. The experiments were performed as in (B) except that p53−/−;RNPC1+/+and p53−/−;RNPC1−/− MEFs were used. (E) Ectopic expression of RNPC1 shortens the half-life of MDM2 transcript. p53−/−HCT116 cells were uninduced or induced to express RNPC1 for 24 h, followed by treatment with 5 μg/ml actinomycin D for various times. Total RNAs were isolated and then subjected to quantitative RT-PCR analysis. The level of MDM2 transcript was normalized to that of GAPDH control and the relative half-life of MDM2 was calculated. Data were presented as Mean±S.D. from triplicate samples. (F) Knockout of RNPC1 prolongs the half-life of MDM2 transcript in p53-null MEFs. The experiment was performed as in (E) except that p53−/−;RNPC1+/+ andp53−/−;RNPC1−/− MEFs were used.