Figure 4.

Increased IRES-dependent expression of p53 and Δ40p53 and downstream activation of target mRNAs under glucose deprivation is SMAR1 dependent. (a–c) Western blot analysis of H1299-NS- and H1299-S3-cell extracts transfected with pGFP-hp-p53-5'UTR-cDNA plasmid (14A in schematic; a), pGFP-hp-p53-5'UTR(A135T)-cDNA plasmid (14A-M2 in schematic; b) or ΔIRES-Δ40p53 plasmid (c) that were glucose starved (−G) for 8 h before harvesting, compared with unstarved (+G) cells. (d–f) Dual-luciferase reporter assay for pRp53(1–134)F (IRES1, d), pRp53(135–251)F (IRES2, e) and pRp53(1–251)F (IRES1+2, f) bicistronic constructs transfected in H1299-NS and H1299-S3 cells that were glucose starved (−G) for 8 h before harvesting, compared with unstarved (+G) cells. Fold change in IRES activity (Fluc/Rluc) shown with this ratio unit normalized for unstarved H1299-NS cells, n=6. (g) Quantitative PCR for levels of p53-target mRNAs p21/Cip1 (CDK-interacting protein1), Mdm2 (murine double minute 2) and TIGAR (TP53-induced glycolysis and apoptosis regulator), assayed after 0, 8 and 30 h of glucose deprivation of H1299-NS and H1299-S3 cells transfected with pGFP-hp-p53-5'UTR-cDNA (14A) plasmid, n=3. Representative immunoblots depicting SMAR1, p53 and Δ40p53 levels are shown