Supplementary Data.
Materials and Methods:
Materials:
SP600125 (JNK) and U0126 (MEK1/2) inhibitors were from Merck VWR (Lutterworth, UK).
Nilotinib and BEZ235 were obtained from Novartis (Camberley, UK). AZD6244 was obtained
from Astra Zeneca (Macclesfield, UK). Lapatinib was obtained from Glaxo Smith Kline
(Uxbridge, UK). Sorafenib was obtained from Bayer (Newbury, UK). The Rac1 G-LISA kit
(#BK128) was from Tebu-Bio (Peterborough, UK).
Methods:
Rac1 G-LISA.
Rac-GTP was measured using the Rac1 G-LISA activation assay. Schwannoma cells were
infected with GFP and Sox10/GFP expressing adenoviruses for 48 hours and 0.5-0.8µg of
lysate applied to the Rac G-LISA following the manufacturer’s instructions. Mean
absorbance was measured at 490nm and values for Sox10 normalised against control. Pvalues were calculated using a student T-Test and are denoted on figures for <0.05 as *,
<0.01 as ** and <0.005 as ***.
Adhesion Assays.
500 mouse Schwann cells were plated onto coverslips coated with 4µg/ml laminin and
incubated for 20 minutes at 37°C. Cells were then washed twice with 1ml phosphate
buffered saline and fixed for 10 minutes in 4% paraformaldehyde. Following a 30 minute
stain with Hoechst dye, every cell nuclei per coverslip was counted. Cell counts were
performed for 4 coverslips per point, and the experiment performed three times with control
and Sox10 null mouse Schwann cells. P-values were calculated using a student T-Test and
are denoted on figures for <0.05 as *, <0.01 as ** and <0.005 as ***.
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MAP Kinase and Tyrosine Kinase Inhibitor Differentiation Experiments.
All inhibitors were added 30 minutes in defined medium (DM) before cAMP (1mM)
stimulation as follows; AZD6244 (1µM), BEZ235 (100nM), Lapatinib (3µM), Nilotinib (0.5µM),
Sorafenib (0.5µM), U0126 (10µM) and SP600125 (10µM). For Lapatinib, Niolotinib and
Sorafenib, half the original concentration was re-added after 24 hours. For all inhibitor
experiments, only a single inhibitor compound was used in each experiment presented.
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Figures
Figure S1
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Figure S2
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Figure S3
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Figure S4
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Figure S5
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Figure S6
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Figure Legends.
Figure S1: Merlin null schwannoma cells do not induce Krox-20 or periaxin in
response to cyclic AMP (cAMP). Control (NF2 +/+) and Merlin null (NF2-/-) cells
were treated with 1mM cAMP for 24, 48 and 72h and percentage Krox-20 positive
nuclei (A) or periaxin positive cells (B) counted at each timepoint after cAMP addition.
DM: control cells in defined medium alone.
Figure S2: Treatment of human schwannoma cells with MAP kinase or tyrosine
kinase inhibitors does not restore Krox-20 induction in response to cyclic AMP.
Human schwannoma cells were pre-treated with AZD6244 (1M), BEZ235 (100nM),
Lapatinib (3M), Nilotinib (0.5M) or Sorafenib (0.5M) prior to addition of 1mM
cAMP. Cells were stained for Krox-20 48h after addition of cAMP. Rat Schwann cells
(SC) were used as a positive control for Krox-20 induction by cAMP.
Figure S3: Phosphorylation of p65 subunit of NFB (Ser276) and CREB (Ser133)
following cAMP addition are unchanged in Merlin null schwannoma cells.
Timecourses of percentage positive phospho-NFB (A) and phospho-CREB (B) after
addition of 1mM cAMP to control Schwann (NF2+/+) and Merlin null (NF2-/-)
schwannoma cells.
Figure S4: Combined counts of percentage Sox10 positive Schwann cells from
analysis of cryostat sections from control nerve (NF2+/+) and schwannoma tumours
(NF2-/-). Counts are averages from 3 normal nerves and 6 schwannoma tumours.
Figure S5: Loss of Sox10 alone from Schwann cells increases cell adhesion. Cell
adhesion assay for control GFP and Sox10 null (CRE) infected mouse Schwann
cells.
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Figure S6: Re-expression of Sox10 in human schwannoma cells reduces Rac-GTP
loading. Shown are results for Rac G-LISA analysis for human schwannoma cells
48h after infection with GFP control or GFP/Sox10 (Sox10) adenoviruses.
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