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. 2015 Jun 18;43(13):6568–6578. doi: 10.1093/nar/gkv617

Figure 4.

Figure 4.

3′-Tailed mirtrons targeted against VEGFA result in functional knockdown of VEGF-A in a cell culture model of hypoxia. (A) HEK293 cells were transfected with 500 ng of NAD/TMirts and 100 ng of the matched psiCheck2.2 targets (T1 and T2 contain concatamers of the target sequences in the 3′ UTR). Cells were harvested two days after transfection. (B) HEK293 cells were transfected with 500 ng of mirtrons/NAD and subjected to hypoxic conditions 1 day after transfection for 24 h before harvesting. qRT-PCR of VEGF was performed using RNA harvested from the cells and normalized against GAPDH. ELISA was performed on the cell culture supernatant; total human VEGF secreted in 500 μl of media, as measured by ELISA, is indicated. (C) Sequences of TMirts compared to their miRNA equivalents. Mutations made are indicated by substitutions of the nucleotides within the boxes. (D) HEK293 cells were transfected with 500 ng of NAD/TMirts mutants / miRNA mimics and 100 ng of the matched psiCheck2.2 VEGFA T1. Cells were harvested two days after transfection. (E) High-throughput sequencing of small RNAs from HEK293 cells transfected with 500 ng of the relevant plasmids. *P < 0.05, **P < 0.01, ***P < 0.001 versus NAD control, n = 3 biological replicates for each experiment unless otherwise stated. Error bars reflect standard deviation.