20µl of the assay solution was added to the cells after 48 hours and incubated for one hour before measurement of absorbance at 490nm carried out in a microplate reader. Briefly, 10,000 transduced normal and patient bone marrow cells were seeded to 96-well plate and treated with L-leucine as previously described. Cell proliferation was measured by CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega) according to manufacturer’s instruction. Viable cell counts were determined by trypan blue exclusion on day 2, 4, and 7 after L-leucine addition. Cell expansion assays Cultured cells at day 7 from the two MDS patients with del(5q) were seeded into 96-well plates (10,000 cells per 0.1ml) and treated with L-leucine as previously described. The filters were then dissolved in scintillation cocktail to measure radioactivity on a Beckman LS6500 counter. Proteins were recovered using spin-x-UF Concentrators (Sigma-Aldrich) with polyethersulfone membranes (PES). The solution was incubated for 30 minutes on After centrifugation at 4✬, cells were resuspended in 50µl of bovine serum albumin (1mg/ml Sigma-Aldrich), and then 450µl of 10% Trichloroacetic acid was added.
After 10 minute incubation at 37✬, ice-cold PBS was added to the cells. After centrifugation, cells were resuspended in IDMEM medium containing only -L-Leucine 10♜i/ml (Perkin Elmer Cambridge). Evaluation of translation efficiency Cells from healthy controls expressing RPS14 shRNAs or cells from MDS patients with del(5q) were evaluated 4 days after treatment with L-leucine and compared to untreated cells viable cells were counted, washed twice in leucine free IDMEM medium and incubated for 15 minutes in the same medium. Each sample was run in triplicate and the expression ratios were calculated using the ΔΔCT method.4 Western blot Western blot was performed using the Invitrogen NuPage Novex 4–12% Bis-Tris Gels as previously described.5 Anti-RPS14 antibody (M01 Abnova) and anti-beta actin antibody HRP (Abcam) at 1:500 and 1:20,000 dilutions were used for detection. Pre-developed TaqMan Assays were used (Assays-on-Demand, Applied Biosystems, Foster City, CA, USA) and reactions were run on a LightCycler 480 Real-Time PCR System (Roche Diagnostics, Lewes, UK). The β2microglobulin gene was used to normalize for differences in input cDNA. Real-time quantitative PCR The expression levels of RPS14 were determined using real-time quantitative PCR. After incubation with lentivirus for 24 hours, infected cells were selected with 1.2µg/ml puromycin. CD34+ cells were infected with lentivirus in the presence of 8µg/ml polybrene 24 hours after being thawed out. Lentivirus was produced in 293T cells as previously described3 except Lentiviral Packaging Mix (Sigma Aldrich) and TurboFect in vitro Transfection Reagent (Fermentas) were used instead according to manufacturer’s instruction. An shRNA sequence that does not target human genes (“scramble”) was used as experimental control. Lentivirus production and transduction Short hairpin RNA (shRNA) sequences targeting RPS14 and cloned into a pLKO.1 vector were obtained from Sigma Aldrich (TRCN0000008641 and TRCN0000008644). Medium, including cytokines and L-leucine as above, was replenished every second day to maintain the same cell concentration. L-leucine (Sigma Aldrich) at a concentration of 600µg/ml was added at day 7 for 4 days.
On day 7, erythropoietin (Roche, Basel, Switzerland) at 2 U/ml was added to the medium.
CD34+ cells from MDS patients with del(5q) and from healthy controls were cultured as previously described.1,2 Briefly, CD34+ cells were cultured for 14 days in Iscove's Modified Dulbecco's Media (Sigma) supplemented with 15% BIT9500 serum substitute (StemCell Technologies) plus 10 ng/ml IL-3, 10 ng/ml IL-6, and 25 ng/ml stem cell factor (PeproTech). Mononuclear cells were purified by Histopaque (Sigma-Aldrich, Gillingham, UK) density gradient centrifugation, labelled with CD34 MicroBeads and CD34+ cells were isolated using MACS magnetic cell separation columns (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s recommendations. Bone marrow samples were obtained and CD34+ cells isolated from two MDS patients with del(5q). Materials and Methods Cell culture and L-leucine treatment CD34+ cells from bone marrow of healthy controls were purchased from Lonza.