Figure 5.

Longer 3′-UTR isoforms dictate PTEN protein dosage. (A) Schematic showing all 12 shRNAs (shRNA 3′-UTR) used to specifically knockdown the expression of all 3′-UTR isoforms longer than 300 nt without affecting the expression of the short ones, 53 and 300 nt. (B) mRNA expression for Pten long (all isoforms longer than 300 nt) and each specific 3′-UTR isoform, Pten 3.3, Pten 5.5/6.1 and Pten 300, was quantified by qRT-PCR in NMuMG stable cell lines. Data are presented as Pten mRNA fold change for each 3′-UTR shRNA compared to the control shRNA (shc002). (C) The effect of each shRNA 3′-UTR on PTEN protein level was analysed by western blot with α-Tubulin as a loading control. The Odyssey imaging system (LiCOR) was used to quantify PTEN fold change level relative to control shRNA. (D) Pearson’s correlation analysis was performed between the mRNA expression of each Pten 3′-UTR isoform (B) and the corresponding PTEN protein level (C). Pearson’s coefficient (r) values are indicated on the graph with their corresponding P-value (****P ≤ 0.0001). Data shown in (A–C) are means ± standard deviation of four independent biological experiments. In (D), linear regression and 95% confidence interval are presented.