Figure 3. Liposomal small-interfering RNA (siRNA) efficacy in FLASH mice.

(a) FLASH mouse embryonic fibroblasts (MEFSs) were treated with either 10, 30, 90, or 270 nmol/l of luciferase-targeted siRNA (si-Luc), 270 nmol/l of siRNA-targeting factor VII (si-FVII), or left untreated. Bioluminescence imaging (BLI) and cell viability assays were performed 72 hours after treatment. Y-axis represents bioluminescence as photon flux (photons/s) normalized to cell viability. Significant differences between si-Luc and nontreated MEFS are denoted by asterisks; *P < 0.05, **P < 0.01, ***P < 0.001. Error bars represent mean + SEM (n = 3). (b) FLASH mice were injected with either si-Luc or si-FVII and ex vivo BLI was performed on the liver and lung tissue 72 hours after injection. Scale bar represents photons/s/cm²/sr. (c) Luciferase expression levels were quantified using quantitative reverse transcriptase-PCR and an in vitro luciferase activity assay from the liver and lungs of si-Luc and si-FVII-treated FLASH mice. Y-axis represents RNA and protein levels as a percent of si-FVII-treated control mice. Error bars represent mean + SEM (n = 3). Significant differences between si-Luc- and si-FVII-treated animals are denoted by asterisks; *P < 0.05, **P < 0.01, ***P < 0.001. (d) Linear relationship between luciferase and total protein concentration from the liver of a FLASH mouse. Y-axis represents photon flux (photons/s). FLASH, firefly luciferase activated systemically in homozygotes.