br Vector construction Total RNA

Vector construction Total RNA at a concentration of 0.05µg/µL was reverse transcribed with a cDNA cycle kit (Invitrogen, Karlsruhe, Germany) using oligo dT primers and AMV reverse transcriptase. Amplification of the complete cathepsin L coding sequence was by PCR with 500ng cDNA using 0.5µM each of the primers “CL for NheI” (5′-aca cag gtt tta aaa cat gaa tcc tac a-3′) and “CL rev BamHI” (5′-agc tac ccc act gtg tga gct ggt gga-3′), 0.125mM each of dNTPs, and 0.4 U Phusion DNA polymerase (Finnzymes, Espoo, Finland). By this cloning strategy, restriction sites for NheI and BamHI were introduced and the stop codon was removed, thus eventually yielding the sequence for a chimeric protein with the full-length cathepsin L sequence covalently connected to the EGFP tag by a spacer peptide linker. The final PCR product and pEGFP-N1 plasmid were each digested with NheI and BamHI (both, MBI, St. Leon-Rot, Germany). The restricted cDNA was ligated into the linearized vector using T4-DNA ligase (MBI) in the presence of ATP-containing reaction buffer (MBI) at 4°C overnight, followed by ligase inactivation at 65°C for 10min. The ligated plasmids were transformed into competent E. coli JM 109, and kanamycin-resistant clones were used for isolation of vector cDNA (Qiagen, Hilden, Germany), which was eluted and stored in 10mM Tris–HCl at pH 8.0. Control digests of the isolated plasmids were performed with NheI and BamHI, each used at 0.25U/µg plasmid DNA, for insert restriction, and additionally EcoRI with a restriction within the insert was used (all, MBI) before analyzing on 1% agarose gels by electrophoresis. Finally, sequencing using the primers “CMV-Profor” (5′-aaa tgg gcg gta ggc gtg-3′) and “EGFP-Nrev” (5′-cgt cgc cgt cca gct c-3′) confirmed the correct product (Sequiserve, Vaterstetten, Germany).
Transfection of HCT116 cells The human colorectal carcinoma HCT116 cell line was purchased from ATCC (Teddington, Middlesex, UK). The protein phosphatase inhibitor were cultured at 37°C in a 5.0% CO2-atmosphere (Heraeus Instruments GmbH, Osterode, Germany) in RPMI-1640 medium (Biowhittaker™) supplemented with 10% fetal calf serum (Lonza, Verviers, Belgium). Transfection with pEGFP-N1 and phCL-EGFP was performed using X-treme GENE HP DNA transfection reagent (Roche Diagnostics, Mannheim, Germany) following the manufacturer׳s protocol.
MTT assays Cell proliferation rates and metabolic activity levels of transfected HCT116 cells were examined by a colorimetric assay in which the yellow 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) is reduced to form purple formazan crystals by intracellular NAD(P)H-oxidoreductase. The assays were performed in duplicates and repeated at least twice as previously described [8], and the reaction product was quantified at 595nm with a Tecan GENios Reader (Tecan Deutschland GmbH, Crailsheim, Germany).
Lysate preparation, SDS-PAGE, and immunoblotting Transfected HCT116 cells were used 24h post-transfection, and whole cell lysates were prepared in PBS containing 0.2% Triton X-100 as described before [9], normalized to equal amounts of protein and loaded onto 12.5% SDS-polyacrylamide gels (GE Healthcare, 80-1255-53) along with a PAGE ruler pre-stained protein ladder (Fermentas). After transfer onto nitrocellulose, blots were incubated with goat anti-human cathepsin L (1:500, Neuromics GT15049) and rabbit anti-human β-tubulin (1:1000, Abcam 6046-100) with HRP-coupled secondary antibodies used for subsequent visualization by enhanced chemi-luminescence onto CL-XPosure film (Pierce, through Perbio Science Europe, Bonn, Germany).
Results Cathepsin L has been shown to be mis-trafficked in HCT116 cells and to enter the nucleus as an unexpected scene of potential action [1]. Nuclear cathepsin L is suggested to involve in regulation of cell cycle progression of HCT116 cells by accelerating S phase [1]. Moreover, the vector phCL-EGFP coding for EGFP-tagged cathepsin L was expressed in HCT116 cells and sorted to the nucleus [1, Fig. 7D′].