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. 2005 Dec 7;33(21):6884–6894. doi: 10.1093/nar/gki1000

Figure 5.

Figure 5

PTB stimulates HIF-1α IRES-mediated translation. (A) Effect of PTB on HIF-1α IRES-driven expression in a bicistronic reporter assay. HEK293T cells were transfected with the bicistronic reporter plasmid Di-HIF or Di-PITSLRE, together with different amounts of the plasmid for PTB expression (pcPTB). The transfectants were lysed 24 h later and the luciferase activities were measured. The bars represent the average (n = 3) ± SD of the calculated ratio between Renilla and firefly luciferase activity. The results are representative of three independent experiments. (B) Overexpression of PTB does not affect the integrity of the bicistronic mRNA. HEK293T cells were transfected with the pSV-sport vector (lane 1) or with the Di-HIF vector in combination with either the empty pCAGGS vector (lane 2) or the PTB-containing pCAGGS expression vector pcPTB (lane 3). RNA was prepared 24 h later and analyzed by northern blotting using a radiolabeled probe directed against the coding sequence of Renilla luciferase. (C) Translation of the second cistron is initiated from an intact dicistronic mRNA. HEK293T cells were transfected with the bicistronic reporter plasmid Di-HIF, in combination with either an empty expression vector or pcPTB and in combination with siRNAs not directed against any known cellular RNA (si none, white bars), or against the Firefly luciferase mRNA (si Fluc, black bars). Cells were lysed 24 h later, and luciferase activities were measured. Bars represent the average (n = 2) ± SD of the calculated ratio between luciferase and β-galactosidase activities. (D) Effect of PTB on expression controlled by HIF-1α 5′-UTR in a monocistronic reporter assay. pSV-HIF-Rluc or pSV-Rluc were cotransfected with pSV-Fluc and different concentrations of pcPTB in HEK293T cells. The cells were lysed 24 h later and the luciferase activities were measured. The bars represent the average (n = 3) ± SD of the calculated ratio between Renilla luciferase and firefly luciferase. The results are representative of three independent experiments.