Figure 3

Structure and knockdown efficacy of shRNA and miRNA hairpin constructs targeting luciferase and Apolipoprotein B100 (ApoB). (a b c) Predicted stem-loop structures of shRNA and miRNA targeting luciferase: shLuc or miLuc and ApoB: shApoB or miApoB with guide strand highlighted in grey. shRNA structure is described in Figure 1. miApoB consists of pri-mir-155 precursor sequence, where the mature mir-155 sequence was replaced with the target sequence for luciferase or ApoB. ApoB1 and ApoB2 target different sequences in the ApoB gene. (d) Luciferase knockdown by CMV-shLuc and CMV-miLuc. Renilla and Firefly luciferase were measured two days post-transfection with 100 ng shRNA or miRNA expressing plasmid and 2,5 ng Firefly luciferase and 0,5 ng Renilla luciferase. H1-shLuc was used as a positive control. shScr and miScr served as negative controls and were set at 100%. Firefly luciferase expression was normalized to Renilla luciferase expression. Data are represented as mean values ± SD from three independent experiments analyzed with the factor correction method [19] (e) Knockdown of Luc-ApoB1 reporter, containing in its 3’ UTR ApoB1 target sequence, by CMV-shApoB1 and CMV-miApoB1. Experimental setup was as described in (d) H1-shApoB1 was used as a positive control. (f) Knockdown of Luc-ApoB2 reporter, containing in its 3’ UTR ApoB2 target sequence, by CMV-shApoB2 and CMV-miApoB2. Experimental setup was as described in (d) with the exception that in this Renilla luciferase contained target sequence for ApoB2 and its expression was normalized to Firefly luciferase expression H1-shApoB2 was used as a positive control. **p < 0.01 versus negative control (One-way ANOVA test with Bonferroni post test).