PAGE 3 OF 8 Nucleic Acids Research, 2005, Vol. 33, No. 22 e190 Figure 1. QD/siRNA complexes allow sorting of gene silencing in cell populations. (A) Schematic representation of cells co-transfected with QDs and siRNA and analyzed for intracellular fluorescence by flow cytometry. Histograms depict fluorescence distributions of control murine fibroblast cells, Lmna siRNA-treated cells and Lmna siRNA/QD-treated cells. FACS was used to gate and sort the high 10% (H) fluorescence and low 10% (L) fluorescence of each distribution. L and H point to gates for the siRNA only histogram. L+ and H+ indicate gates for the siRNA/QD histogram. (B) Representative western blot of Lamin A/C protein expression levels in sorted cells with b-actin as loading control. Control lanes are protein from cells mock-transfected with liposome reagent only and sorted (L and H). The absence of QDs is indicated by a minus sign ( ) and the presence of QDs is indicate by a plus sign (+). (C) Band densitometry analysis of western blots from replicate experiments. Error bars represent standard error of the mean (n ¼ 3). ***P < 0.001 (One-way ANOVA). MetaMorph Image Analysis software (Universal Imaging) and normalized to loading control (b-actin) bands. Primary antibodies used were polyclonal lamin A/C anti- body (Cell Signaling) at 1:1000 dilution in blocking solution and polyclonal b-actin antibody (Cell Signaling) at 1:750 dilu- tion. T-cad primary antibody was a gift from Barbara Ranscht (University of California, San Diego) (33). Secondary anti- body was goat anti-rabbit IgG-horseradish peroxidase (HRP) (Santa Cruz Biotechnology) at 1:7500 dilution. Blots were probed simultaneously for lamin A/C protein (70 kDa and 28 kDa) and b-actin protein (45 kDa); after detection, select blots were re-probed for T-cad (95 kDa). Immunofluorescence staining Sorted and unsorted cells intended for lamin nuclear protein immunostaining were seeded on to Collagen-I coated glass coverslips. Coverslips with attached cells were washed twice in cold phosphate-buffered saline (PBS, Gibco) and ﬁxed in 4% paraformaldehyde at room temperature. After three brief PBS washes, cells were permeabilized with 0.2% Triton X for 10 min at room temperature and washed again. The cells were blocked with 10% goat serum for 30 min at 37 C, incubated in primary antibody (1:100 Lamin A anti- body, Santa Cruz Biotechnology) for 90 min at 37 C, washed three times with 0.05% Triton X, incubated in secondary anti- body (1:250 AlexaFluor 594 chicken anti-rabbit IgG antibody, Molecular Probes) for 1 h at room temperature, and washed a ﬁnal three times. Antibody dilutions were performed in 1% BSA in PBS. Coverslips were mounted on to glass slides using Vectashield anti-fade medium (Vector Laboratories). Finally, nuclear staining was visualized and documented by phase contrast microscopy or epiﬂuorescence (Nikon Ellipse TE200 inverted ﬂuorescence microscope and CoolSnap-HQ Digital CCD Camera). Hepatocyte/fibroblast co-cultures Hepatocytes were isolated from 2 to 3 month old adult female Lewis rats (Charles River Laboratories) and puriﬁed as described previously (34,35). Fresh, isolated hepatocytes were seeded at a density of 2.5 · 105 cells per well, in 17 mm wells adsorbed with 0.13 mg/ml Collagen-I. Cultures were maintained at 37 C, 5% CO2 in hepatocyte medium consisting of DMEM with high glucose, 10% FBS, 0.5 U/ml insulin, 7 ng/ml glucagons, 7.5 mg/ml hydrocortisone, 10 U/ml