Materials and Methods
Cell culture and L-leucine treatment
CD34+ cells from bone marrow of healthy controls were purchased from Lonza. Bone
marrow samples were obtained and CD34+ cells isolated from two MDS patients with
del(5q). 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. 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). On day 7,
erythropoietin (Roche, Basel, Switzerland) at 2 U/ml was added to the medium. L-leucine
(Sigma Aldrich) at a concentration of 600µg/ml was added at day 7 for 4 days. Medium,
including cytokines and L-leucine as above, was replenished every second day to maintain
the same cell concentration.
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). An shRNA
sequence that does not target human genes (“scramble”) was used as experimental control.
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. CD34+ cells were infected with lentivirus in
the presence of 8µg/ml polybrene 24 hours after being thawed out. After incubation with
lentivirus for 24 hours, infected cells were selected with 1.2µg/ml puromycin.
Real-time quantitative PCR
The expression levels of RPS14 were determined using real-time quantitative PCR. The β2microglobulin gene was used to normalize for differences in input cDNA. 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). 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.
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. After centrifugation, cells were resuspended in IDMEM
medium containing only [3H]-L-Leucine 10µCi/ml (Perkin Elmer; Cambridge). After 10
minute incubation at 37ºC, ice-cold PBS was added to the cells. After centrifugation at 4ºC,
cells were resuspended in 50µl of bovine serum albumin (1mg/ml; Sigma-Aldrich), and then
450µl of 10% Trichloroacetic acid was added. The solution was incubated for 30 minutes on
ice. Proteins were recovered using spin-x-UF Concentrators (Sigma-Aldrich) with
polyethersulfone membranes (PES). The filters were then dissolved in scintillation cocktail to
measure radioactivity on a Beckman LS6500 counter.
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. Viable cell
counts were determined by trypan blue exclusion on day 2, 4, and 7 after L-leucine addition.
Cell proliferation was measured by CellTiter 96 AQueous One Solution Cell Proliferation
Assay (Promega) according to manufacturer’s instruction. Briefly, 10,000 transduced normal
and patient bone marrow cells were seeded to 96-well plate and treated with L-leucine as
previously described. 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.
Colony forming cell assays
Colony forming cell assays were performed using Human Methylcellulose Complete Media
(R&D Systems) according to manufacturer’s instruction. Assessment of the number of BFUE colonies formed from erythroid progenitors was performed after 14 days in culture.
Flow cytometry
To evaluate erythroid differentiation, cells were incubated for 30 minutes on ice with antiCD36-PE (BD Bioscience Pharmingen), anti-CD71-APC (eBioscience) and anti-CD235aFITC (BioLegend) antibodies before staining with DAPI (Sigma Aldrich) to gate out dead
cells. Assessment of total p53 protein level by intracellular staining was performed as
previously described.6 To perform apoptosis assay, cells resuspended in Annexin V buffer
were stained with Annexin V-FITC antibody for 20 minutes in the dark according to
manufacturer’s instruction (BD Bioscience Pharmingen). 1µg/ml propidium iodide was
added to the cells immediately before analysis. To perform cell cycle analysis, cells were
fixed with ice-cold absolute ethanol for 30 minutes before incubation with 40µg/ml
propidium iodide and 10µg/ml RNase for one hour at 37°C. Flow cytometry was performed
on a BD LSRII (BD Bioscience) and the data were analysed using FlowJo software version
7.6.4.
Gene expression profiling
Gene expression profiling was performed using Affymetrix HG-U133 Plus2.0 arrays
(approximately 39,000 genes) as previously described.7
Statistical analysis
Statistical significance was determined by student t-test.
References
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