MCF-7-Luc cells were prepared by plating

cells in a 6-well plate 24 h prior to each experiment. For transfection, each lipoplex of 200 pmol Luc siRNA, Luc-siRNA-Chol, Cont siRNA or Cont siRNA-Chol Nutlin-3 chemical structure was diluted in 2 ml of medium supplemented with 10% FBS and then the mixture was added into the cells. Lipofectamine RNAiMax lipoplex (Invitrogen Corp.) was prepared according to the manufacturer’s protocol. Forty-eight hours after the transfection, luciferase activity was measured as counts per s (cps)/µg protein using the luciferase assay system (Pica gene luciferase assay kit; Toyo Ink Mfg. Co., Ltd.) and BCA reagent (Pierce, Rockford, IL, USA), as previously reported [11]. Agglutination Selleck Dorsomorphin assay was performed according to the method described in a previous report [5]. Briefly, erythrocytes were collected from mouse blood at 4 °C by centrifugation at 300g for 3 min and resuspended

in PBS as a 2% (v/v) stock suspension of erythrocytes. The anionic polymer-coated lipoplexes of 2 µg of Cont siRNA or siRNA-Chol were added to 100 µL of erythrocyte suspension and then incubated for 15 min at 37 °C. The sample was placed on a glass plate and agglutination was observed by microscopy. All animal experiments were performed with approval from the Institutional Animal Care and Use Committee of Hoshi University. Cationic and anionic polymer-coated lipoplexes of 50 µg of Cy5.5-siRNA or Cy5.5-siRNA-Chol were intravenously administered via lateral tail veins into female BALB/c mice

(7 weeks of age; Sankyo Lab. Service Corp., Tokyo, Japan). One hour after injection, the mice were sacrificed, and the tissues were frozen on dry ice and sliced at 16 µm. The localization of Cy5.5-siRNA was examined using an Eclipse TS100-F microscope (Nikon, Tokyo, Japan). Anionic polymer-coated lipoplexes of 50 µg of Cont siRNA-Chol or ApoB siRNA-Chol were intravenously administered via lateral tail veins into mice. At 24 h post-injection, mice were fasted for 24 h. At 48 h post-injection, mice were sacrificed by cervical 6-phosphogluconolactonase dislocation and the liver was removed for analysis. Total RNA was isolated from the liver using the NucleoSpin RNA II (Macherey-Nagel, Germany). Mouse ApoB cDNA was amplified using the primers ApoB-FW, 5′-TTCCAGCCATGGGCAACTTTACCT-3′, and ApoB-RW, 5′-TACTGCAGGGCGTCAGTGACAAAT-3′, as previously reported [ 12]. Mouse β-actin cDNA was amplified using the primers β-actin-FW, 5′-TGTGATGGTGGGAATGGGTCAG-3′, and β-actin-RW, 5′-TTTGATGTCACGCACGATTTCC-3′, as previously reported [ 13]. Quantitative RT-PCR was performed with the iCycler MyiQ detection system (Bio-Rad Laboratories, Hercules, CA, USA) and the SYBR Green I assay (iQ™ SYBER Green Supermix, Bio-Rad Laboratories), as previously described [13]. Samples were run in triplicate and the mRNA expression levels of ApoB were normalized to the amount of β-actin mRNA in the same sample.