Gene pulser 2 electroporation apparatus

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).

Using the pAL5000-TOPO vector, an EGFP-expressing Mycobacterium-Escherichia coli shuttle vector was generated as previous study (51 (link)). Briefly, the EGFP gene was amplified from the pIRES2-EGFP vector (Clontech, Mountain View, CA, USA; Cat No., 6029–1), and the hsp65 promoter gene was amplified from genomic DNA of M. bovis BCG. The EGFP gene with the hsp65 promoter was amplified from the pMyong2-EGFP^h vector and ligated into pAL5000-TOPO using HindIII and BamHI restriction sites. The hsp65promoter and EGFP gene were amplified by overlapping PCR. To generate three different types of recombinant strains expressing EGFP (Asan 50594, M. smegmatis and BCG), the pAL5000-TOPO-EGFP plasmid was electroporated into competent bacteria (Asan 50594, M. smegmatis and BCG) using the Gene Pulser II electroporation apparatus (Bio-Rad, Hercules, CA, USA). Transformants were selected on Middlebrook 7H10 medium (Difco Laboratories, Detroit, MI, USA) (52 (link)) supplemented with OADC and containing 100 μg/ml kanamycin. Typically, colonies of transformants were selected from the plates, transferred into 7H9 broth medium (Difco Laboratories, Detroit, MI, USA) supplemented with 0.5% glycerol, 0.05% Tween-80, 10% ADC and 100 μg/ml kanamycin and cultured for 3~5 days. The growth rate of the recombinant mycobacteria strains was determined by measuring the medium OD at 600 nm.