Transfection of MDA-MB-231 Human Breast Cancer Cells with Bola/siRNA Complexes

The siRNA duplexes in the in silico, in vitro, in vivo, and ex vivo experiments were designed to silence EGFP as described in our previous study.32 siRNA transfections were performed with the human breast cancer cell line MDA-MB-231 (that either express or do not express EGFP) using vesicles made from the bolas GLH-19, GLH-20, or a mixture of the two. Upon resuspension in water, the bola vesicles (1 mg/mL) were mixed with siRNAs (100-fold the desired final concentration) at a 1:1 volume ratio. Upon 30 min of incubation at room temperature, the bola/siRNA complexes were diluted 50-fold in Opti-MEM prior to addition to the cells for transfection. Upon a 4-h incubation at 37°C, the media were replaced by regular DMEM for 24 h (transfection efficiency) or 72 h (silencing efficiency).

To assess the silencing efficiency, cells were imaged after the transfection with a Nikon 200 TE inverted microscope (Nikon, Melville, NJ, USA). To visualize the uptake of the bola/siRNA particles, experiments were performed with a LSM 710 confocal microscope (Carl Zeiss) and RNAs were labeled with Al546. The cells were fixed with 4% paraformaldehyde and imaged (transfection efficiency) or permeabilized with 0.2% Triton X-100 for 20 min in order to label the early endosome-associated protein EEA1 (with primary [Cell Signaling Technology, Danvers, MA, USA] and Al488-conjugated secondary [Molecular Probes, Eugene, OR, USA] antibodies), and Nikon 200 TE-Al488 imaging was performed with the 488-nm line of an argon laser while Al546 imaging was obtained through excitation with a DPSS 561 laser.

For statistical analysis of the flow cytometry experiments, at least 30,000 MDA-MB-231 cells (Cell Biolabs, San Diego, CA, USA; with or without EGFP) as well as A375 (BRAF^V600E) and HMCB (BRAF^WT) cells (gifts from Dr. Deborah K. Morrison, NCI-Frederick) were analyzed by FACS analysis with a FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

A375 and HMCB cells were plated at 10⁵ cells/35-mm dish and transfected with 100 nM (final concentration) siRNA-Al488, BRAF^V600E siRNA (5′-GCUACAGAGAAAUCUCGAUUU-3′), or a non-targeting siRNA pool (Thermo Fisher Scientific, D-001810-10-05). Briefly, the siRNAs were incubated with GLHs for 30 min at 25°C, protected from light. After incubation, the mixture was diluted in 1 mL of Opti-MEM (Thermo Fisher Scientific, #31985088) and directly added to cells previously rinsed with PBS. After 4 h, the mixture was replaced with regular serum-containing media. For siRNA uptake assays, cells were trypsinized after 24 h and analyzed by FACS. For knockdown experiments, whole-cell lysates were generated after 48 h using radioimmunoprecipitation assay (RIPA) buffer (10 mM Tris [pH 7.4], 150 mM NaCl, 1 mM EDTA, 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100). Antibodies used for immunoblotting were anti-BRAF (Santa Cruz, sc-5284) and anti-β-actin (Santa Cruz, sc-1616-R).