Cell culture and treatments
K562 cells were cultured in RPMI 1640 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 10% v/v foetal bovine serum (Invitrogen, Gibco) and maintained at 37°C with 5% CO2. For proteasome inhibition, cells were treated with 10 μM MG132 for the final 18 hours of culturing. For autophagy inhibition, cells were treated in culture with either 10 mM 3-methyladenine from day 3-day 7 post transfection or 1 μM bafilomycin A1 for 48 hours day 5-day 7 post transfection.
The shRNA vector used was a modified pcDNA3.1 vector (pcDNA3.1-H1) developed by Heiko Lickert  (kind gift from Heiner Schrewe) in which the CMV promoter has been replaced by the human RNAse P RNA H1 promoter . CSN2 and CSN5 silencing sequences were selected using a siRNA design tool available on http://www1.qiagen.com/products/genesilencing/customsiRNA/siRNA designer.aspx and cloned into the Asp718 and XbaI restriction enzyme sites of pcDNA3.1-H1. The target sequences are as follows:
CSN2 knockdown 5'AAGCGGCATTAAGCAGTTTCC3'
CSN5 knockdown – 5'AAGGGCTACAAACCTCCTGAT3'
shRNA scramble control – 5'AAGCGGGATTCAGTAGTTACG3'
Transfections and cell sorting
Transfection efficiencies in K562 cells vary between 20–50%. Therefore, to allow enrichment of transfected cells, 5 × 106 K562 cells were electroporated in Nucleofector kit V solution (Amaxa) using a Nucleofector I (Amaxa) and file T16, with 5 μg pMACS Kk.II and 10 μg of the relevant knockdown pcDNA3.1-H1 plasmid according to manufacturer guidelines. The pMACS Kk.II produces a truncated murine MHC class I cell surface protein, H-2Kk, which lacks the cytoplasmic domain and is transiently expressed on the cell surface of transfected cells between 6 and 48 hours post-transfection. Transfected cells were sorted 24 hours post transfection using anti-H-2Kk antibody conjugated to magnetic beads, MACS MS columns and a MACS magnet (Miltenyi Biotec) according to manufacturer instructions. Post sorting, cells were set at 3 × 105/ml daily and cells harvested for protein and mRNA analysis as indicated in results.
Thymidine incorporation assay
2 × 104 cells were pulsed with 2 μCi/ml 3H-thymidine (Amersham) for the final 18 hours of culture leading up to each time point. Samples were transferred to a filter mat (Wallac) using a Skatron cell harvester (Skatron Instruments) and read using a beta-plate scintillation counter (Skatron Instruments).
Immunofluorescence and Jenner-Giemsa staining
Cytospins were made with 5 × 104 cells in 80 μl, using a Shandon cytospin 3 (Shandon). For immunofluorescence staining, cytospins were fixed in 4% paraformaldehyde and stained using anti-β-tubulin antibody (Sigma, 1/500 dilution) followed by FITC labelled secondary antibody (Jackson Laboratories, 1/500 dilution). DNA was counterstained using Hoescht 33342 (Sigma, 1/1000 dilution). All reagents were diluted in PBS and slides mounted using Mowiol (6 g glycerol, 2.4 g Moviol-4-88 (Sigma), 12 ml 0.2 M Tris HCl pH8.5, anti-fade crystal (Sigma), 6 ml distilled water). Slides were viewed using an Axioskop2 microscope (Zeiss) and images captured with a Q-imaging 12-bit QICAM (Media Cybernetics) and Openlab software (Improvision).
For Jenner-Giemsa staining, cytospins were air-dried, methanol fixed and stained; First with Jenner staining solution (VWR, UK) diluted 1/3 in 1 mM sodium phosphate buffer pH5.6 (5 mins) and second with Giemsa stain (VWR, UK) diluted 1/20 in 1 mM sodium phosphate buffer pH5.6 (10 mins). Slides were dried and then mounted onto coverslips using DePex (VWR, UK). Slides were viewed with an Olympus BX40 microscope (Olympus) and images captured using an Olympus Chameleon digital SLR (Olympus).
Staining of autophagosomes
For visualisation of autophagic vacuoles, 5 × 104 cells were incubated with 0.05 mM monodansylcadaverine (MDC, Sigma) in 0.5 ml PBS for 10 minutes at 37°C. Cells were washed four times with PBS, cytospins made as above and cells viewed immediately using a Leica DMIRE2 system.
Fluorescence flow cytometry
For Annexin V labelling, 1 × 105 cells were stained using Annexin V-FITC Apoptosis Detection Kit I (BD Biosciences) according to manufacturer instructions, and staining analysed within 1 hour by flow cytometry. For cell cycle analysis 1 × 105 cells were resuspended in cell cycle buffer (10 μg/ml propidium iodide, 0.1 mM sodium chloride, 1% Triton X100) and samples analysed within 24 hours by flow cytometry. All staining was analysed using a FACS Calibur (Becton Dickinson) and the data evaluated using Cell Quest Pro software (Becton Dickinson).
Western blot analysis
Whole cell lysates were prepared using RIPA buffer (1% v/v NP40, 0.5% w/v sodium deoxycholate, 0.1% w/v 10% SDS, in distilled water) and protein quantified using the Dc protein assay according to manufacturer instructions (Bio-Rad). Forty micrograms of protein were boiled for 10 minutes in 1× SDS gel loading buffer (15.6 mM Tris HCl pH6.8, 6.25% v/v glycerol, 0.5% SDS, 1.25% v/v 2-mercaptoethanol, Bromophenol Blue, in distilled water). Proteins were separated by SDS-PAGE and transferred to PVDF membrane (Millipore). For western blot analysis, the following antibodies were used at 1:1000 dilution: CSN2 (Bethyl), CSN5 (Bethyl), Cul-1 (Zymed), Skp2 (Zymed), p27 (Santa Cruz), caspase-9 (Cell Signalling), LC-3 (Novus Biologicals) and β-actin (Sigma). Proteins recognized by these antibodies were detected using ant-mouse (Sigma, 1/1000 dilution) or anti-rabbit (Pierce, 1/1000 dilution) HRP conjugated secondary antibody followed by enhanced chemiluminescence (SuperSignal West Pico Chemiluminescent Substrate, Pierce) and autoradiography (Kodak X-Omat LS film, Sigma). Quantitative analysis of western blots was carried out using ImageJ software http://rsb.info.nih.gov/ij/download.html and protein levels normalized by comparison to β-actin signals on the same membrane.
2-Dimensional gel analysis
Native protein extracts were obtained from 2.5 × 105 cells by resuspending cells in 50 μl mild lysis buffer (25% 4× NativePAGE sample buffer (Invitrogen), 1% digitonin, 10% 10× protease inhibitor, in distilled water). Extracts were separated out in the first dimension using a NativePAGE Novex Bis-Tris Gel System (Invitrogen) according to manufacturer instructions. The gel was then cut into individual lanes, proteins denatured by incubation in 1× SDS gel loading buffer and resolved in the second dimension by electrophoresis through 12.5% SDS-polyacrylamide gels. Proteins were transferred to PVDF membrane (Millipore) and immunoblotting performed as above.
Quantitative real-time PCR analysis (QRT-PCR)
RNA was extracted using the Qiagen RNeasy kit according to manufacturer instructions and cDNA generated using 1 μg RNA, random hexamers (Promega) and Superscript II reverse transcriptase (Invitrogen). Quantitative real-time PCR was carried out using either TAQMAN or SYBR-Green based assays. For TAQMAN assays, QRT-PCR was carried out in duplicate 20 μl reactions containing 1× qPCR Mastermix Plus (Eurogentec), 20–40 ng cDNA, 18 pmoles each primer and 2.5 pmoles FAM/TAMRA dual labeled probes. For SYBR-Green assays, QRT-PCR was carried out in duplicate 25 μl reactions containing 1× Sensimix (Quantace), 20–40 ng cDNA, 9 pmoles each primer, 1× SYBR-Green solution (Quantace), 4 mM MgCl2 and 0.5 units UNG (Quantace). QRT-PCR was carried out on an ABI Prism 7000 sequence detector (Applied Biosystems). The following primers (Sigma Genosys) and FAM/TAMRA labeled probes (Eurogentec) were used:
CSN2, 5'-CCTCATCCACTGATTATGGGAGT-3' (forward),
CSN5, 5'-ATATCCGCAGGGAAAG-3' (forward),
5'- TGGCGCCTTTAGGACATACCCAAAGG-3' (probe);
Skp2, 5'-CGCTGCCCACGATCATTT-3' (forward),
Cdc4, 5'-ACGACGCCGAATTACATCTGT-3' (forward),
β-Trcp, 5'-GAGGCATTGCCTGTTTGCA-3' (forward)
18S, 5'-GCCGCTAGAGGTGAAATTCTTG-3' (forward),
Preoptimised primers and probes to 18S ribosomal RNA were used as internal standards in TAQMAN QRT-PCR (Applied Biosystems). Cycle threshold (Ct) values were obtained graphically for test genes and 18S internal standards. ΔCt values were calculated by subtracting 18S Ct from test gene Ct, and average ΔCt values obtained from duplicates. Relative mRNA levels were determined by subtraction of mock transfection ΔCt values from shVC/shCSN2/shCSN5 ΔCt values to give a ΔΔCt value and conversion through 2-ΔΔCt.