Cell Culture
p16/p19-/- EGFRviii NSCs were generated from various genetic
backgrounds using identical techniques. Cells were grown and passaged as
previously describred (Bachoo et al 2002, Zheng et al 2008).
EGFRviii
neurospheres were injected intracranially within the 6 passages.
For differentiation or transfection experiments of NSCs, cells were seeded
on poly-L-ornithine/laminin coated surfaces as previously described (Zheng et al
2008). Cells were differentiated by withdrawal of FGF/EGF and allowed to
differentiate over 6 days. Media was replaced every other day.
Human U251 and HEK 293T cells were grown in Dulbeco's modified
Eagle's medium (DMEM) supplemented with 10% fetal calf serum and
penicillin/streptomicyn (Invitrogen, Carlsbad, CA) in a 5% CO 2 humidified
incubator at 37 oC.
Glioma tissue from p53-/-;NF1-/- mouse genetic mosaic model was
dissociated into single cells by papain method. giNSCs with oligodendrocyte
precursor cells properties were enriched by anti-PDGFRa immunopanning
method(Dugas et al 2006). The enriched cells were cultured at 37˚C, 10% CO2 in
Neurobasal medium (Invitrogen) containing human transferrin (100 µg/ml),
bovine serum albumin (100 µg/ml), putrescine (16 µg/ml), progesterone (60
ng/ml), sodium selenite (40 ng/ml), glutamine (2mM), sodium pyruvate (1mM),
Penicillin-Streptomycin (100 U each), 1:50 dilution B-27 supplement (all from
Invitrogen). The growth factors added were EGF (50 ng/ml) and PDGF-AA (10
ng/ml). Cells were cultured on poly-D-lysine (Sigma) coated tissue culture dishes
or flasks.
To passage cells, when glioma cells reached confluence or near
confluence, media was removed and a small volume of Trypsin-EDTA solution
(Invitrogen 25300-054) was added to the cells until the cells started to become
non-adherent. An equal volume of trypsin inhibitor solution (200 µg/ml Trypsin
Inhibitor, Roche 109878) plus 0.4% BSA in DPBS without Ca ++ or Mg++
(Invitrogen) was used to remove cells by gentle trituration.
Cells were then
centrifuged at 220 x g for 10 minutes, the pellet rinsed with DPBS without Ca ++ or
Mg++, spun down again, and resuspended in growth media described above.
giNSCs (005 cells) were cultured in N2-supplemented (Invitrogen)
DME:Ham’s F12 (Omega Scientific) medium containing 20 ng/ml fibroblast
growth factor-2 (PeproTech), 20 ng/ml epidermal growth factor (Promega) and 50
ug/ml heparin (Sigma).
Lentiviral Preparation and Transduction
Lentivirus production and titering were carried out according to protocols
from Tronolab (http://tronolab.epfl.ch). Different clones of p16/p19-/- EGFRviii
neurospheres and giNSCs (005 cells) were transduced with miR-128 expressing
lentivirus (also expressing GFP) with MOIs of 1/3/9. We isolated pure miR128/GFP expressing cells by FACS.
RNA Isolation and Real-Time PCR Analysis
RNA was extracted with the miRVana Isolation Kit (Ambion). The miRNA
levels were assayed with the Taqman probes and primer sets in an Applied
Biosystems PRISM 7900HT Fast Real-Time PCR System (Applied Biosystems)
as previously described(Neveu et al 2010). For mRNA expression analysis, we
followed a previously published procedure(Papagiannakopoulos et al 2008). All
reactions
were
performed
according
to
the
manufacturer’s
protocols.
Normalizations for mRNA and miRNA RealTime PCR were performed using the
Ct of GAPDH and RNU6B respectively.
Luciferase Reporter Transfection and Dual Luciferase Assay
HEK 293T cell 3’UTR experiments were performed as previously
described(Xu et al 2009). We used 30 pmol of pre-miR miRNA precursor
molecules (Ambion) or LNA antisense-oligos (Exiqon) with 2 μl of Lipofectamine
2000. For NSCs 3’UTR experiments, 1 x 105 cells were seeded on 24-well plates
coated with fibronectin/poly-L-Ornithine.
800ng of the pMIR-Report vector
(Ambion) 3’UTR constructs and 250 ng of the transfection control Renilla vector
phRLTK (Promega) were transfected with 1.5 μl of Lipofectamine LTX
(Invitrogen) and 2.5 μl of Plus Reagent. HEK and NSC lysates were harvested 24
hr after transfection, and reporter activity was measured with the Dual Luciferase
Assay (Promega). Luciferase constructs with 3’ UTR sequences
The 3’UTRs of EGFR and PDGFRA were PCR amplified from NSC
genomic DNA, cloned into pMiR-Report (Ambion) downstream of the firefly
luciferase gene and verified by sequencing. Mutagenesis of predicted targeted
sites was achieved using Quikchange site-directed mutagenesis kit (Stratagene)
following instructions.
FACS and Flow Cytometry Analysis for Markers
For isolation of subpopulation of NSCs that expressed Green Fluorescent
Protein (GFP) from lentiviral infection, cells were sorted on BD FACS Aria cell
sorter Rrewith 100μm nozzle and according to instructions from facility
instrument technicians.
For differentiation experiments, cells were collected after differentiation
and fixed with 70% Ethanol ice-cold at -20Co overnight.
For staining a cell
suspension (1–5 × 105 cells) was incubated at room temp for 2hr in 100μl
staining buffer (1x PBS+1%BSA+0.1%Triton-X) containing primary antibody
against a marker.
secondary
After washing, the cell suspension was incubated with
PE-conjugated
Goat
anti-Mouse
or
anti-rabbit
(Jackson
ImmunoResearch) for 30min at room temp. Detection was performed using
Guava EasyCyte Flow Cytometer (Guava Technologies).
Cell Death and Apoptosis Assays
U251 cells were transfected with control pre-miR or pre-miR-128 (Ambion)
and 24h or 48h post-transfection cells were collected and stained with Guava
Viacount reagent (Guava, Millipore). Stained cells were analyzed using Guava
EasyCyte Flow Cytometer (Guava Technologies).
Statistical Analysis
Statistical Analysis was either determined by ANOVA or Student’s t-test.
For Kaplan-Meier survival curves Gehan-Breslow-Wilcoxon and Log-rank
(Mantel-Cox) Tests were performed. Statistical Significance is displayed as
P<0.05 (*), P<0.01 (**), P<0.001 (***).