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Supplementary Figures Legends
Supplementary Figure 1. (A) U87 and U87-EGFRvIII (5 x 104) cells were treated with IL1β (0-100 ng/mL) for 24 h and IL-8 secretion was determined by ELISA (for details see
Methodology below). Values were normalised to cell viability (MTT) and expressed as foldincrease (mean ± SEM) compared to untreated U87 cells. (B-C) U87 and U87-EGFRvIII cells
were incubated with fluorescein-labelled p38 MAPK inhibitor SB203580 (10μM) for 30 and
60 min. Cells were lysed (B) and fluorescence intensity of 100 μg whole cell lysate was
measured (for details see Methodology below). Alternatively, (C) cells were trypsinised,
resuspended and fluorescence intensity was analysed by flow cytometry. (D) U87 and U87EGFRvIII cells were starved for 2 h, pre-treated with SB203580 (SB, 10μM) for 60 min and
incubated with IL-1β (10 ng/mL) for 24 h. IL-6 in supernatants (extracellular IL-6) and cell
lysates (intracellular IL-6) was determined by ELISA and normalised to total protein
concentration (BCA assay). Data are expressed relative to IL-6 levels in IL-1β treated cells
and represent the mean ± SEM from 3 independent experiments performed in duplicates. (E)
U87 and U87-EGFRvIII (3 x 105) cells were starved for 2 h, pre-treated with 10 μM
SB203580 (SB) and treated with IL-1β (10 ng/mL) for 24 h. IL-8 mRNA was determined by
RT-PCR and normalised to 18s mRNA as described (1-3). Data are expressed as fold-increase
compared to untreated cells and represent the mean ± SEM from at least 3 independent
experiments performed in duplicates. (**P < 0.01, ***P < 0.001, 1-way ANOVA followed by
Newman-Keuls post-test using Prism 5 GraphPad Software).
Supplementary Figure 2. (A-C) U87, U87-EGFRvIII and U87-EGFRwt (4 x 105) cells were
starved for 2 h and treated with IL-1β (10 ng/mL) for 0-120 min. Whole cell lysates were
prepared and protein concentration was determined (for details see Methodology below). 25
μg cellular protein were subjected to Western blot analysis as described (4) and analysed for
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p38 MAPK phosphorylation (p-p38), total p38 MAPK (p38), phosphorylated MK2 (p-MK2),
total MK2 (MK2), phosphorylated Hsp27 (p-Hsp27) and total Hsp27 (Hsp27) as indicated.
Representative blots of three independent experiments are shown.
Supplementary Figure 3. (A) U87 and U87-EGFRvIII (4 x 105) cells were starved for 2 h
and treated with IL-1β (10 ng/mL) for 2 h. Whole cell lysates from U87 (lanes 1-2) and U87EGFRvIII (lanes 3-4) were analysed for protein concentration. 25 μg cellular protein were
subjected to Western blot analysis as described (4) and analysed for CREB and ATF-1
phosphorylation (p-CREB, p-ATF-1), respectively, and total CREB (CREB) as indicated.
Representative blots of three independent experiments are shown. (B) U87 and U87EGFRvIII (3x105) were transfected with 1 μg IL-6-luc651 and 1 μg pSV-β-galactosidase for
24 h. Cells were then starved for 2 h, pre-treated with 10 μM SB203580 for 30 min, followed
by IL-1β (10 ng/mL) stimulation for 24 h. Cells were harvested, and luciferase and βgalactosidase activities determined as described (1).
Supplementary Figure 4. (A) Whole cell lysates from U87 and U87-EGFRvIII were
analysed for protein concentration and 20 – 40 μg cellular protein were subjected to Western
blot analysis as described (4). Immunoblots were analysed for total p38 MAPK (p38),
phosphorylated and total MK2 (p-MK2 and MK2). Protein expression was determined and
normalised against β-tubulin. Representative titration blots and quantification of relative p38
MAPK (p38) and MK2 protein expression from three independent experiments are shown.
(B) U87 cells were transfected with HuR siRNA or scrambled siRNA as a negative control
for 72 h (for details see Methodology below). Whole cell lysates were subjected to Western
blot analysis under non-reducing conditions to detect HuR monomer (37 kD) and dimer (75
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kD). Rabbit polyclonal anti-HuR (#20694, Santa Cruz) and mouse monoclonal anti-HuR
(#5261, Santa Cruz) antibodies were used as indicated. β-tubulin served as a loading control.
Supplementary Figure 5. (A-B) U87 and U87-EGFRvIII (1 x 107) cells were activated with
IL-1β (10 ng/mL) for 0-30 min. Nuclear and cytoplasmic fractions were prepared and
analysed by Western blotting for phosphorylated and total MK2 (p-MK2, MK2),
phosphorylated and total Cdk1 (p-Cdk1, Cdk1), phosphorylated and total HuR (pHuR(S202),
HuR), lamin A/C and β-tubulin (for details see Methodology below). Relative p-MK2 and
HuR levels were quantified and normalised to lamin A/C (nuclear fractions) and β-tubulin
(cytosolic fractions). Relative p-Cdk1 and p-HuR(S202) levels were quantified and
normalised to total Cdk1 and total HuR, respectively. Representative blots and data (mean ±
SEM) from two independent experiments are shown. (C) U87 and U87-EGFRvIII (1x107)
were starved for 2 h, pre-treated with SB203580 (10 μM) for 60 min and activated with IL-1β
(10 ng/mL) for 8 h. Protein concentration of nuclear and cytosolic fractions was determined
and 25 μg of nuclear protein fraction was subjected to Western blot analysis. Nuclear
distribution of HuR, lamin A/C and β-tubulin is shown (representative for three independent
experiments).
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Supplementary Table 1.
U87 and U87-EGFRvIII (5 x 104) cells were starved for 2 h, pre-treated with compounds (10
μM) inhibiting p38 MAPK (SB203580), MEK1/2 (PD98059) or JNK (SP600125) for 60 min
and treated with IL-1β or TNFα (10 ng/mL) for 24 h. Secretion of IL-6 and IL-8 was
determined by ELISA, respectively, and normalised to cell viability as above. See
Methodology below for further details.
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Methodology
Reagents and antibodies. Recombinant IL-1β and TNFα were obtained from R&D Systems.
All chemicals including inhibitors for p38 MAPK (SB203580), JNK (SP600125), MEK1/2
(PD098059) were obtained from Sigma Aldrich. p38 MAPK inhibitor MW-01-SRM-2-069A
(069A) was developed in our laboratory and has been described in detail (5). MK2 inhibitors
sc-48 (#204048), sc-38 (#203138), mouse monoclonal antibody against EGFR (#365829),
rabbit polyclonal antibody against HuR (#20694), mouse monoclonal antibody against HuR
(#5261) and rabbit polyclonal antibody against CRM1 (#5595) were from Santa Cruz. Rabbit
monoclonal antibodies against phosphorylated p38 MAPK (#9215), β-tubulin (#2128),
phosphorylated CREB (#9197), total CREB (#9198); rabbit polyclonal antibodies against
total p38 MAPK (#9212), MK2 (#3042), phosphorylated Hsp27 (#2401), lamin A/C (#2032),
total Cdk1 (#9112), phosphorylated Cdk1 (Tyr15) (#9111); mouse monoclonal antibody
against total Hsp27 (#2402); the secondary anti-mouse IgG, HRP-linked monoclonal antibody
(#7076) and the secondary anti-rabbit IgG, HRP-linked monoclonal antibody (#7074) were
obtained from Cell Signaling Technology. pHuR(S202) antibody was kindly provided by
Myriam Gorospe (National Institutes of Health, Baltimore, Maryland, USA).
Cell culture. Primary human astrocytes were purified from 14 to 19 week old aborted
foetuses, collected after therapeutic termination after obtaining informed consent (approved
by the Human Ethics Committee of the University of Sydney (HREC 2013/131) and
University of New South Wales (HREC08284) as described (6). U87 and U87-EGFRvIII
glioblastoma cells were provided by their laboratory of origin (7) and tested for EGFR
mutation and stable expression by Western blotting (Figure 3C) and immunostaining. The
cumulative culture length of all cell lines was less than 3 months. GBM cell lines (U87, U87EGFRvIII) and primary human astrocytes were cultured in DMEM and RPMI-1640
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(Invitrogen), respectively, supplemented with 10 % (v/v) foetal bovine serum (FBS,
Invitrogen) and antibiotic-antimycotic solution (Gibco) at 37 C and 5 % CO2.
Cytokine expression. Cytokine production was determined by ELISA using BD OptEIA
Human IL-6 and IL-8 kits (BD Biosciences), following manufacturer’s instructions.
Western blotting. Cells were harvested in lysis buffer (20 mM Tris-HCl, pH 7.5, 2 mM
EDTA, 100 mM NaCl, 5 mM MgCl2, 1% (v/v) Triton X-100, 5 mM NaF, 10% (v/v) glycerol,
0.5% (v/v) 2-mercaptoethanol, 0.1 mM Na3VO4 and protease inhibitors (Sigma Aldrich
P8340). After centrifugation at 10.000 x g the protein concentration of the cleared cell lysate
was determined using a Bicinchoninic Acid (BCA) Protein Assay Kit (Pierce), following
manufacturer’s instructions. Cell lysates were separated by 10 – 12.5 % SDS-PAGE and
transferred to Immobilion-P (Millipore) as described (4). Proteins were detected using their
specific primary antibodies, respectively, followed by goat anti-rabbit horseradish
peroxidase–conjugated secondary antibody or horse anti-mouse horseradish peroxidase–
conjugated secondary antibody, and detection using the SuperSignal West Pico
Chemiluminescent Substrate (Thermo Scientific) and the ChemiDoc MP System (Bio-Rad).
HuR detection by Western blotting. In order to detect HuR monomer and dimer in nuclear,
cytosolic fractions or whole cell lysates, subcellular fractions or whole cell lysates were
prepared in 4x Bolt Lithium Dodecyl Sulphate (LDS) sample buffer (Life Technologies)
without a reducing agent. Subcellular fractions or whole cell lysates were separated by 10 –
12.5 % SDS-PAGE and transferred to Immobilion-P (Millipore) as described (4). Proteins
were detected using freshly prepared rabbit polyclonal antibody against HuR (#20694, Santa
Cruz, overnight at 4 °C at a 1:500 dilution), followed by goat anti-rabbit horseradish
peroxidase–conjugated secondary antibody, and detection using the SuperSignal West Pico
Chemiluminescent Substrate (Thermo Scientific) and the ChemiDoc MP System (Bio-Rad).
In some experiments (Figure 3 and Suppl Figure 5), HuR immunoblots were run under the
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MK2-HuR pathway in glioblastoma inflammation
same conditions, but DTT (50 mM) was added to the LDS buffer. The identity of HuR
monomer and dimer was verified in lysates from HuR-depleted cells (see HuR knockdown
studies below).
Subcellular fractionation. Subcellular fractionation of U87, U87-EGFRvIII and primary
astrocytes (1 x 107) was performed using NE-PER Nuclear and Cytoplasmic Extraction
Reagents (Thermo Scientific) following manufacturer’s instructions. The extracts were stored
at -80 ˚C prior to Western blot analysis. The purity of nuclear and cytoplasmic fractions was
verified using anti-lamin A/C and β-tubulin antibodies.
HuR knockdown studies. 1 x 106 U87 and U87-EGFRvIII cells were transfected with 75
pmol HuR siRNA targeting human HuR from position 325 to 345 (Invitrogen #4390824,
sense
5’-ACUUAUUCGGGAUAAAGUATT-3’,
antisense
5’-
UACUUUAUCCCGAAUAAGUTT-3’) and Lipofectamine RNAiMAX (Invitrogen #13778)
following manufacturer’s protocol. After 72 h, cells were stimulated with IL-1β (10 ng/mL)
for further 24 h. Supernatants were collected for IL-6 quantification and cell lysates were
subjected to Western blot analysis. Scrambled siRNA (Invitrogen #4390843) served as a
negative control.
Reverse transcriptase-polymerase chain reaction (RT-PCR). Total RNA was extracted using
the RNea
The integrity of
total RNA was verified on agarose gels and 1 μg RNA was reverse transcribed using the
Fermentas RevertAidTM First strand cDNA Synthesis Kit following manufacturer’ protocol.
RT-PCR to amplify cDNA fragments encoding IL-6 and IL-8 were performed and quantified
as described (2, 3).
IL-6 mRNA stability assay. U87 and U87-EGFRvIII (4 x 105) were starved for 2 h, pretreated with MK2 inhibitor sc-48 (10 μM) for 60 min and activated with IL-1β (10 ng/mL) for
4 h. Cells were then washed and incubated with actinomycin D (5 μg/mL) to inhibit further
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MK2-HuR pathway in glioblastoma inflammation
transcription. Cells were harvested after 0, 0.5, 1, 2 and 4 hours of incubation with
actinomycin D. IL-6 mRNA was quantified by real-time PCR as described (2). Results are
presented as % IL-6 mRNA remaining compared with mRNA expression after 4 h of IL-1β
treatment. Decay constants (k) were calculated by nonlinear regression (one phase
exponential decay) of the % IL-6 mRNA remaining versus time after actinomycin D (3) using
Prism 5 (GraphPad Software, San Diego, CA).
Luciferase activity assays. 3 x 105 U87 and U87-EGFRvIII cells were transfected with 1 μg
pGL3 luciferase reporter containing a 651-bp fragment of the human IL-6 gene promoter
(pIL-6-luc 651) together with 1μg pSV-β-Galactosidase (Promega) and 4 μL Lipofectamine®
2000 (Invitrogen) following manufacturer’s protocol. After 48 h, cells were lysed in 150 μL
reporter lysis buffer (Promega, #E4030). Luciferase activity (Promega, #E4030) was
measured at 490 nm and normalized to β-galactosidase activity (Promega, #E2000) as
described (1).
SB203580 internalisation. U87 and U87-EGFRvIII (2 x 105) cells were starved for 2 h and
fluorescein-labeled SB203580 (10 μM) was added for 30 and 60 min, respectively. Cells were
washed twice with PBS and lysed in 100 μL lysis buffer (20 mM Tris-HCl, pH 7.5, 2 mM
EDTA, 100 mM NaCl, 5 mM MgCl2, 1% (v/v) Triton X-100, 5 mM NaF, 10% (v/v) glycerol,
0.5% (v/v) 2-mercaptoethanol, 0.1 mM Na3VO4 and protease inhibitors). Cells lysates were
cleared by centrifugation for 5 min at 10,000 x g and fluorescence intensity of duplicate
samples was measured at 382 nm using the Novostar plate reader (BMG Labtech).
Fluorescence relative units (FRU) were normalised to total cellular protein. Alternatively,
SB203580 internalisation was determined by flow cytometry. 4 x 105 cells were incubated
with 10 μM fluorescein-labelled SB203580 for 30 and 60 min as above. Cells were
trypsinised and resuspended in PBS with 0.1% (w/v) BSA. Identical cell numbers (1x105)
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MK2-HuR pathway in glioblastoma inflammation
were analysed with the FACSCalibur flow cytometer (Becton Dickinson) using CellQuestPro
Software. Data analysis was performed using FlowJo Software.
Immunohistochemistry. Formalin-fixed, paraffin-embedded tissues from 205 brain tumour
patients with grade II - IV astrocytomas who underwent surgery (Unit of Neurosurgery,
Tampere University Hospital, Tampere, Finland) during 1983 – 2001 were used (approved by
Ethical Review Board of Tampere University Hospital, Tampere, Finland, Dnro R07042;
National Authority for Medicolegal Affairs of Finland, Dnro 1502/04/046/07). Whole tissue
sections of histologically normal brain tissue obtained from the University of
Sydney/Neuropathology Tumour and Tissue Bank (approved by University of Sydney HREC
2013/131) served as control (n = 6). Following dewaxing and rehydration, tissue section
(thickness, 5 μm; area 0.28 mm2) peroxidase activity was blocked by incubation in 0.5%
H2O2 in methanol. Tissue was then treated for 30 min in citrate buffer (pH 6.0) at 121C for
antigen retrieval. Activated MK2 (p-MK2) was detected using rabbit polyclonal antibody
recognising pT334-MK2 (#3007, Cell Signaling Technology). The EnvisionTM+Dual Link
(Dako) was applied for 20 min, followed by Dako liquid DAB+chromogen according to
manufacturer’s recommendations. The tissue sections were counterstained with hematoxylin.
p-MK2 immunoreactivity in TMA samples was evaluated by two pathologists (JH and MB)
using a consultation microscope (Zeiss Axioscope). Immunoreactivity was scored negative
(<5% positive cells) or positive (>5% positive cells). The positive samples were assigned
score 1 (weak staining), score 2 (moderate staining) or score 3 (strong staining) based on the
highest intensity observed in the sample (8). Examples of these intensities are in the Figure
4E.
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MK2-HuR pathway in glioblastoma inflammation
Statistical Analysis.
ELISA data analysis was performed using Prism 5 (GraphPad Software, San Diego, CA) and
represents the mean ± SEM from at least three independent experiments. Statistical analysis
of ELISA data was performed using one-way ANOVA, followed by the Newman-Keuls
multiple comparison test (GraphPad Software). P values <0.05 were considered as statistically
significant. Immunoblots were analysed using Image J. IC50 values were calculated by nonlinear regression analysis from at least three independent experiments (each treatment data
point performed in duplicate) with Excel software.
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MK2-HuR pathway in glioblastoma inflammation
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