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. 2020 Feb 11;21(4):1206. doi: 10.3390/ijms21041206

Figure 3.

Figure 3

Evaluation of a putative internal ribosome entry site (IRES) in the 5′ UTR of ACLY mRNA. (A) HepG2 cells were co-transfected with pGL3prom, pGL3-ACLY or pGL3S1a constructs together with the control plasmid pcDNA3.1/HisB/lacZ. At 24 h after transfection, FL activity was measured and normalized with respect to β-galactosidase activity. Values are means ± S.D. (n = 6) and are reported as a percentage of FL activity determined in cells transfected with the empty vector pGL3 prom. (pGL3 prom vs. pGL3-ACLY *p ≤ 0.05). (B) HepG2 cells were co-transfected with pR-S1a-F or pR-ACLY-F constructs, together with pcDNA3.1/HisB/lacZ used for the normalization of transfection efficiency. RL and FL activities, normalized to β-galactosidase activity, are reported as the fold change relative to those determined in cells transfected with the control vector pRF. Values are means ± S.D., (n = 6). (pRF vs. pR-S1a-F and pR-ACLY-F * p ≤ 0.01). (C) HepG2 cells were co-transfected with pRF, pHpR-S1a-F or pHpR-ACLY-F, together with pcDNA3.1/HisB/lacZ used for the normalization of transfection efficiency. RL and FL activities, normalized to the β-galactosidase activity, were reported as the fold change relative to those determined in cells transfected with the control vector pRF. Values are means ± S.D., (n = 6). (Right panel, pRF vs. pHpR-S1a-F and pHpR-ACLY-F * p ≤ 0.05; left panel, pRF vs. pHpR-S1a-F and pHpR-ACLY-F # p ≤ 0.01). (D) The promoterless pRF(-P) or pR-ACLY-F(-P) constructs were co-transfected together with pcDNA3.1/HisB/lacZ into HepG2 cells. At 24 h after transfection, cells were harvested for determination of FL activity, which was normalized to the β-galactosidase activity. Values were reported as the fold change relative to the FL activity measured in HepG2 cells transfected with pRF(-P). Values are means ± S.D., (n = 6).