Figure 4.

LVs encoding the U1-driven miR-agshRNAs produce knockdown of VEGFA and no reduction in viability

(A) A schematic vector map of the pCCL-based lentiviral transfer vector encoding the U1-driven RNAi effectors. U1, U1 snRNA promoter; U1t, U1 terminator box. (B) HEK293 cells were transduced with LVs encoding minimal Vegfa target 12 reporters (see Figure S5A). Then they were transduced with the LVs encoding the U1-driven RNAi effectors, and the Fluc activity was measured after 3 days. Mean Fluc levels (relative light units [RLU]) of triplicates ± SD, normalized to HEK293 cells transduced with the reporter only. See Figure S5B for flow cytometry evaluation. (C) RT-qPCR quantification of the mRNA levels of VEGFA in the 293-hVEGFA cells transduced with the LV encoding the indicated U1-driven RNAi effector. Geometric mean of the PPIA-normalized VEGFA FC relative to the NT control with geometric SD. (D and E) Western blot-assessed VEGFA levels in 293-hVEGFA cells transduced with the LV encoding the U1-driven RNAi effectors, quantification, and representative blot. The intracellular VEGFA levels are presented relative to the total protein content, while the extracellular samples are volume normalized. Mean of triplicates ± SD, normalized to the NT samples. (F) MTT-based cell number assessment of ARPE19 cells 5 days post transduction with the LVs encoding the indicated U1-driven miR451 or miR-agshRNAs. The background-subtracted absorbance at 570 nm relative to the untreated NC is presented. Mean of quintuplicates ± SD. The NT and NC are in triplicates.