Figure 2.

Identification of the IRES activity in the ATF2 5′-UTR. (A) The plasmids pRAF, pRXF, pRNF and pRF were transfected into the cell lines HEK293, Bel7402, HCT-8 and NIH-3T3. The IRES activity was expressed as the ratio of downstream cistron expression to upstream cistron expression (FL/RL). The error bars indicate the SD determined from ≥3 independent experiments performed in triplicate. (B) Schematic representation of the expression cassette of the dual-luciferase bicistronic constructs pRF, pRAF, pRXF and pRNF. (C) The translation of the second cistron is not due to reinitiation or aberrant messenger RNA species. To test the cryptic promoter activity in the ATF2 5′-UTR, the sequence was cloned upstream of the FL reporter, and the SV40 promoter was removed. The empty vector was used as a negative control. The plasmids were transfected into Bel7402 cells. The RL and FL activities were measured after 24 h. The error bars indicate the SD determined from ≥3 independent experiments performed in triplicate. (D) Schematic representation of the expression cassette of the dual-luciferase bicistronic constructs pRF and pRAF in which the promoter was deleted. RL, Renilla luciferase; FL, firefly luciferase; SV, simian virus; ATF2, activating transcription factor 2; NRF, nuclear respiratory factor; XIAP, X-linked inhibitor of apoptosis protein; IRES, internal ribosome entry segment; UTR, untranslated region; SD, standard deviation.