Figure 2.

SMAR1 knockdown inhibits p53 IRES function. (a) Western blot of H1299-cell extracts co-transfected with pGFP-hp-p53-5'UTR-cDNA plasmid (14A, shown in schematic), pSV40-β-galactosidase plasmid (transfection and cap-dependent translation control) and non-targetting (Nsp) or SMAR1 siRNA. Inset shows fold change in β-galactosidase activity in each of the experimental conditions (1–6), n=3. (b) Dual-luciferase reporter assay for relative IRES activity from pRp53(1–251)F plasmid, represented by the Fluc/Rluc ratio, on SMAR1 knockdown in H1299 cells, n=9. Immunoblot in inset shows knockdown of SMAR1 in same lysates. (c) Puromycin-selected H1299 cells with stable non-targetting (NS) or SMAR1 (S3) shRNA expression were transfected with pGFP-hp-p53-5'UTR-cDNA plasmid. Western blot was done 48 h post-transfection. (d) Caspase 3/7 activation assay in H1299 cells following SMAR1 knockdown in the presence of pGFP hp-p53-5'UTR cDNA (14A plasmid). Puro, puromycin (2 μg/ml, 24 h) used as positive control for caspase induction and apoptosis. Experiment performed 48 h post-transfection, n=3. (e) Δ40p53 target 14-3-3σ mRNA levels as measured by qRT-PCR in H1299-NS and H1299-S3 cells transfected with pGFP-hp-p53-5'UTR(A135T)-cDNA (14A-M2) plasmid, n=9. (f) Western blot of H1299-NS- and H1299-S3-cell extracts that were transfected with pGFP-hp-p53-5'UTR-cDNA (14A) or pGFP-hp-p53-5'UTR(A135T)-cDNA (14A-M2) plasmid. Proteasomal degradation inhibitor MG132 (10 μM) was added 1 h before harvesting. Western blot was done on same gel and membrane; results are shown at same exposure