Figure 2.

PKM2 suppression increases p27 levels and cell cycle arrest via HuR-dependent increased p27 mRNA translation. (a) Expression of Renilla and Firefly luciferase genes in lysates from cells from Figure 1aand Supporting Information Figure S1a containing a bicistronic reporter construct in which cap-dependent translation of both genes is driven by the CMV promoter while expression of Firefly can additionally be driven in a cap-independent manner from the internal ribosome entry site in an upstream p27 5’LTR. Quantitative PCR was used to normalize luciferase expression to bicistronic transcript levels. See also Supporting Information Figures S2a–S2b. *, p < .05, n = 3. (b) Control or PKM2 knock-down U87 cells from Figure 1a and Supporting Information Figure S1a were lysed and subjected to sucrose density gradient centrifugation, with subsequent RNA from fractions containing unassembled ribosomal subunits (fractions 2–5) or assembled polyribosomes (fractions 6–10) analyzed for p27 and β-actin mRNA content by quantitative PCR. Each bar represents the fraction of β-actin or p27 mRNA contained in the polysomal fractions in control or PKM2 knock-down cells. See also Supporting Information Figure S2c. Error bars indicate standard deviations, *, p < .05, n = 3. (c–e) Levels of HuR, p27 and β-actin (c), cell cycle distribution (d), and cap-dependent and cap-independent translation of p27 mRNA in cells from Figure 1a also expressing nontargeted (HuR siRNA-) or one of two HuR-targeted (HuR siRNA1 and HuR siRNA2) siRNA. See also Supporting Information Figures S2d–S2f. *, p < .05, n = 3.