GalNAc-Ncoa4-siRNA induces Ncoa4 knockdown in murine hepatoma cells and mouse liver. (A) Ncoa4 expression relative to β-actin (Actb) in total RNA prepared from Hepa1-6 cells harvested 24 hours after treatment with transfection reagent alone (vehicle) or after transfection of a commercial scrambled control siRNA lacking a GalNAc-modification (Ambion Silencer Select Control No. 1 siRNA, cat. #4390843), a commercial murine Ncoa4-targeting siRNA lacking a GalNAc-modification (Ambion Silencer Select Ncoa4 siRNA #s77517), GalNAc-Luc-siRNA (Luc), or GalNAc-Ncoa4-siRNA. siRNAs (10 nM) were delivered with Lipofectamine RNAiMAX to 2.5 × 10⁵ cells per well. n = 6 per group. The mean mRNA expression ratio obtained from vehicle-treated cells was normalized to 1. In the absence of transfection reagent, direct addition of GalNAc-Ncoa4-siRNA to the culture media did not suppress Ncoa4 mRNA (data not shown), consistent with the reported absence of ASGPR expression in Hepa1-6 cells.⁶³  (B) Ncoa4 expression relative to Actb in total RNA prepared from mouse livers harvested 24 hours after injection of vehicle, GalNAc-Luc-siRNA (3 mg/kg), or GalNAc-Ncoa4-siRNA (1, 3, or 5 mg/kg). The mean mRNA ratio obtained from livers of vehicle-treated mice was normalized to 1. n = 5 per group. (C) Ncoa4 expression relative to Rpl19 in total RNA prepared from a panel of mouse tissues harvested 24 hours after injection of either vehicle or GalNAc-Ncoa4-siRNA (5 mg/kg). BM, bone marrow. Expression of Rpl19 (rather than Actb) was used for normalization because of high expression of other actin isoforms in cardiac and skeletal muscle. The mean mRNA ratio obtained from livers of vehicle-treated mice was normalized to 1. n = 5 per group. For all panels, data represent mean ± standard deviation (SD). **P < .01, ***P < .001, and ****P < .0001 by 1-way ANOVA with Tukey’s post hoc test (panels A and B) or by Student t test (panel C).