Siport electroporation buffer

Exponentially growing 2×10⁵ cells were suspended in 75 μl siPORT electroporation buffer (Ambion) containing miR-351 mimic, non-silencing siRNA, miR-351 inhibitor or negative control miRNA inhibitor (final concentration 8×10⁻⁷ M) and introduced into a 0.1-cm gap electroporation cuvette (Bio-Rad). Cells were then electroporated using the Bio-Rad Gene Pulser Xcell at voltage 0.15 kV, pulse length 1,000 μs, and number of pulse 1. After electroporation, cells were plated at 5×10⁴ cells/ml in fresh ES medium in tissue culture flasks. Forty-eight hours after the electroporation, cells were used for further experiments. Transfection efficiencies at higher than 80% were obtained in this electroporation protocol by using the nonsilencing siRNA conjugated with Alexa Fluoro 488 [see ref. [20 (link)] for the conditioning].

The Jurkat, HeLa, DLD-1, MCF-7 and Caco-2 cells were the gifts from P. Tucker, C. Sullivan, and J. Dudley (University of Texas, Austin). DLD-1 and Caco-2 colon carcinoma cell lines were described previously [88] (link), [89] (link). The cell lines were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (Atlanta Biologicals, Norcross, GA). HeLa, DLD-1, Caco-2, or MCF-7 cells were plated in 6-well (8×10⁴ cells per well) or 12-well plates (4×10⁴ cells per well) and transfected after 24 hours using siPORT amine reagent (Ambion, Austin, TX) or Metafectene SI (Biontex-USA, San Diego, CA) with miRNA mimics (Ambion) at a final concentration of 60 nM (Table S1). All cells (including floating cells) were harvested after 48 hours, counted using a hemocytometer, and cell extracts were prepared for Western blot analysis. Trypan blue staining was used to evaluate the number of dead cells. For TRAP analysis cells were harvested 4 hours after transfection. miRNA inhibitors (Ambion) were electroporated into Jurkat cells under 200 V, 1 µF in siPORT electroporation buffer (Ambion) using Gene Pulser II electroporation apparatus (BioRad).

Transfection of Dicer‐targeted or nonsilencing siRNA (siNS) was performed by electroporation as previously described [10, 12, 13]. A set of four siRNAs against Dicer was used, namely Mm_Dicer1_1 (catalog number, SI00979335), Mm_Dicer1_2 (catalog number, SI00979342), Mm_Dicer1_3 (catalog number, SI00979349), and Mm_Dicer1_6 (catalog number, SI02747101). AllStars Negative Control siRNA (catalog number, 1027280) was used as siNS. These siRNAs were obtained from QIAGEN. The Dicer‐targeted siRNA mixture was prepared by combining the Dicer‐targeted four siRNAs. In the 0.1‐cm‐gap cuvette, F28‐7 cells (2 × 10⁵ cells) were suspended in 75 μL siPORT electroporation buffer (Ambion, Austin, TX, USA), containing Dicer‐targeted siRNA mixture or siNS (final concentration: 800 nm). Subsequently, cells were electroporated using the Gene Pulser Xcell (Bio‐Rad, Hercules, CA, USA) at voltage of 0.15 kV, pulse length of 1000 μs, and number of pulse of 1.