Figure 4

CHD1 knockdown enhances cell invasiveness. (a) CHD1 knockdown by two independent short hairpin RNAs (shRNAs), in two different nontumorigenic prostate epithelial cell lines (OPCN2 and RWPE-1) verified by western blot analysis. Percent residual expression (relative to control nontargeting shRNA, and normalized to glyceraldehyde-3-phosphate-dehydrogenase) is indicated. OPCN2 cells were purchased from Asterand (Detroit, MI, USA) and grown in keratinocyte serum-free media (Invitrogen, Grand Island, NY, USA) supplemented with 2% fetal bovine serum (Hyclone, Logan, UT, USA). RWPE-1 cells were obtained directly from the American Type Culture Collection and grown in keratinocyte serum-free media supplemented with 0.05 mg/ml bovine pituitary extract and 5 ng/ml epidermal growth factor (Invitrogen). For RNAi-mediated knockdown, two different GIPZ shRNAs targeting CHD1 (112 969 and 112 972), along with a non-silencing-GIPZ shRNA, were obtained from Open Biosystems (Lafayette, CO, USA). The shRNAs were separately transfected (using Fugene HD reagent; Roche, Indianapolis, IN, USA) into 293TN cells, along with VSVG and pCMVdelta8.91 packaging plasmids, to generate replication-defective lentivirus. Viral supernatant was collected 48 h post transfection and used to infect OPCN2 and RWPE-1 cells. Pooled transductants were selected using 1 μg/μl puromycin for 14 days. Cells were lysed in RIPA buffer, and 30 μg whole-cell lysate was electrophoresed on a 4–15% Tris-HCl polyacrylamide gradient gel. Protein was transferred onto a polyvinylidene fluoride membrane, which was then blocked for 1 h with 5% dry milk in TBS-T buffer, and then incubated overnight with primary antibody at 4 °C. Detection was carried out using a horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence (ECL) kit (GE Healthcare, Piscataway, NJ, USA). Quantification was performed using ImageJ (http://imagej.nih.gov/ij). Antibodies used were anti-CHD1 rabbit polyclonal antibody (NB100-60411, 1:200; Novus biologicals, Littleton, CO, USA), anti-glyceraldehyde-3-phosphate-dehydrogenase (sc-25778, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and horseradish-peroxidase conjugated anti-rabbit IgG (1:10 000; Pierce, Rockford, IL, USA). (b) CHD1 knockdown does not significantly alter cell viability/proliferation, measured by WST-1 assay 1 and 5 days after plating. WST-1 (Roche) is a colorimetric assay of mitochondrial activity in viable cells. (c) CHD1 knockdown promotes cell invasion through Matrigel. Photomicrographs show representative fields of crystal-violet stained traversed cells. *P<0.01; **P<0.01, two-sided Student's t-test. Cell invasion was measured by Boyden chamber assay (BD Biosciences, Bedford, MA, USA). In all, 50 000 cells/24-well insert for OPCN2, or 100 000 cells for RWPE-1, were seeded onto pre-coated filters (8 μM pore size, Matrigel 100 μg/cm²), using a 0.5–10% fetal bovine serum gradient. After 24–48 h, cells traversing the filter were fixed with 10% buffered formalin, stained with crystal violet and manually counted. (d) CHD1 knockdown enhances cell clonogenicity (colony formation on tissue culture plastic) of OPCN2 cells. *P<0.01; **P<0.01, two-sided Student's t-test. Clonogenicity was measured by sparse plating of 600 cells/15-cm plate. After 2 weeks, plates were stained with Giemsa and the number of colonies was counted. All the above assays were done in triplicate (mean±1 s.d. reported), and all experiments were replicated at least once.