siGLO RISC-Free Control siRNA (siGLORNAi), chemically modified to impair processing and uptake by RISC, one-target plus SMART pool siRNAGAPDH and one-target plus SMART pool siRNABcl2 were from Thermo Scientific Dharmacon (GE Healthcare, CO, USA). Peptide synthesis was perfomed by CASLO Laboratory, Lyngby, Denmark. Hoechst Stains and Lipofectamine 2000 were from Invitrogen (CA, USA). Cholera Toxin B Subunit FITC Conjugate was from Sigma (MO, USA).
A20 is a murine cell line derived from a spontaneously arising tumor in an aged BALB/c mouse. It pathologically mimics the characteristics of human diffuse large B cell lymphoma . Cell line was kindly provided by Prof. C. Palmieri (University of Catanzaro, Italy). The cell line was grown in suspension culture with RPMI 1640 medium (GIBCO, CA, USA), supplemented with 10% fetal bovine serum (GIBCO), 50 units/ml penicillin, 50 μg/ml streptomycin and 2 mM L-glutamine at 37 °C in a 5% CO2 atmosphere. NIH/3T3 is a murine embryonic fibroblast cell line. The cell line was provided by CEINGE, Advanced Biotechnologies (Naples, Italy). The cell line was grown in adhesion culture with Dulbecco's Modified Eagle's Medium (GIBCO, CA, USA), supplemented with 10% fetal bovine serum (GIBCO), 50 units/ml penicillin, 50 μg/ml streptomycin and 2 mM L-glutamine at 37°C in a 5% CO2 atmosphere.
Cytofluorimetric analysis and confocal microscopy
Target peptides and random peptides (2 μl, 1 mg/ml, 11.58 μM) plus siGLORNAi (4 μl, 25 μM, 100 pmoles) were incubated at room temperature for 15 minutes at a 1:10 molar ratio in RPMI without FCS and then added to 50 μl of A20 cells at 20 × 106/ml in RPMI 20% FCS. Samples were then incubated at 37°C for 2 hours and 4 hours. After 3 washes in 1× Phosphate Buffered Saline (PBS) (Invitrogen, CA, USA), cells were divided in two aliquots. One aliquot (107/ml) was analysed by flow cytometry whereas the other aliquot was suspended in 2 ml of RPMI and incubated in 6 wells plates overnight and then analysed by flow cytometry. Cells before the cytofluorimetric analysis were permeabilized according to the following procedure: cells were resuspended in 100 μl of 1% paraformaldehyde and incubated at room temperature for 1 hour, washed twice with 200 μl 1× PBS, resuspended in 200 μl 1× PBS, 0.1% Triton X-100 (Sigma, MO, USA), 0.1% sodium cytrate and analyzed at flow cytometry.
The following A20 target-specific peptides were designed based on the peptide sequence reported by Palmieri et al., 2010 . 5R-A20-36-5R, (D-Arg)(L-Arg)4-EYVNCDNLVGNCVI-(L-Arg)4(D-Arg); A20-36-9R; EYVNCDNLVGNCVI-(L-Arg)8(D–Arg); whereas as controls the following random peptides were designed: 5R-RND-5R, (D-Arg)(L-Arg)4-SSAYGSCKGPCSSGVHSI-(L-Arg)4(D-Arg); RND-9R, SSAYGSCKGPCSSGVHSI(L-Arg)8(D-Arg).
For confocal analysis, after incubation of the cells with complexes peptides-siGLORNAi for 4 hours as above reported, about 10,000 cells were plated on small glass disks in a 24 well dishes and let grown for 12 hours. At different times, disks were removed from the wells, washed three times with 1× PBS, stained with membrane stain Cholera Toxin B Subunit FITC Conjugate whereas cell nuclei were stained with Hoechst 33342 and subsequently observed with a Nikon Confocal Microscope C1 equipped with an EZ-C1 Software for data acquisition. Fluorescent image were collected, upon excitation according to dye manufacturing instructions.
siRNAGAPDH and peptide-siRNAGAPDH complex
siRNAGAPDH was used for GAPDH mRNA down-regulation in NIH/3T3. 100 or 250 pmoles of siRNAGAPDH were transfected in the presence of Lipofectamine 2000 (Invitrogen, USA) according to manufacturing protocol. Samples were then incubated at 37°C for 30 and 72 hours. After incubation, cell extracts were then analysed by western blot with anti-GAPDH antibody.
A20-36 target peptides and random peptides plus siRNAGAPDH were incubated according the procedure above reported with A20 cells at 20 × 106/ml in RPMI 20% FCS. After incubation, cells were suspended in 2 ml of RPMI and incubated in 6 wells plates for 48 and 72 hours. Cell extracts were then analysed by western blot with anti-GAPDH antibody.
Western blotting analysis
Cells were collected, washed twice in 1× PBS and resuspended in 20–40 μl of lysis buffer (50 mM Tris–HCl pH 7.4, 1% NP40, 0,25% sodium deoxycholate, 150 mM NaCl, 1 μg/ml aprotinin, leupeptin, pepstatin, 1 mM Na3VO4, 1 mM NaF) for 30 min on ice and centrifuged at 14,000 × g for 20 min at 4°C. Protein concentration was determined according to Lowry et al., 1951 .
Proteins were separated by SDS-PAGE, electro-transferred to PVDF membrane, and incubated over-night at 4 °C with GAPDH monoclonal antibody at dilution of 1:2000. Anti-mouse secondary antibody conjugated with horseradish peroxidase (HRP) (Sigma) was used at a dilution of 1:20,000. The specific protein was visualized by an enhanced chemiluminescence detection reagent (SuperSignal West Pico, Molecular Probes, CA, USA), and exposed to X-ray film. All films were analysed by using Image J software 1.46r.
Total RNA extraction and qRT-PCR were performed as previously described . Briefly, RNA aliquots (500 ng) were reverse transcribed using Random Examers and Superscript III Reverse Transcriptase (Invitrogen), according to the manufacturer's protocol. qRT-PCR was carried out with the iCycler iQ Real-Time detection system (Bio-Rad Laboratories) under the following conditions: 95°C, 1 min; (94°C, 10s; 60°C, 30s) × 40. Primers were as follows: β-actin, forward 5’-GGCACCACACCTTCTACAATGAG-3’ and reverse 5’-GGAGTCCATCACAATGCCTGTGG-3’; GAPDH, forward 5’-GTCAAGGCCGAGAATGGGAAGC-3’ and reverse 5’-AGAAGGGGCGGAGATGATGACC-3’; Bcl2, forward 5’-TCGCAGAGATGTCCAGTCAGC, reverse 5’-CCGGTTCAGGTACTCAGTCATC. GAPDH and Bcl2 expression levels were calculated relative to β-actin mRNA levels as endogenous control by using the formula 2(Ct sample − Ct β-actin) .
Data are reported as average and standard deviation. The statistical significance of differences among groups was evaluated with ANOVA, with the Bonferroni correction as post hoc test, using the software KaleidaGraph v. 4.1. The significance was accepted at the level of p < 0.05.