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FIBROBLAST
GROWTH
FACTOR
RECEPTOR
3….

15
µg
of
protein
from
each
sample
were
subjected
to
10%
SDS-­-
polyacrilamide
gels
and
were
transferred
to
nitrocellulose
membranes.
Thereafter

membranes
were
blocked
and
incubated
overnight
with
anti-­-FGFR3
(1:100)
or

pERK1/2
(1:1,000)
(Santa
Cruz
Biotechnology,
Santa
Cruz,
CA,
USA).
After

washing,
blots
were
incubated
with
peroxidase-­-conjugated
secondary
antibody.

Development
was
performed
using
ECL
system
(Amersham,
Buckinghamshire,

UK).
To
verify
equal
loading,
membranes
were
stripped
in
62.5
mM
Tris-­-HCl
pH

6.8,
2%
SDS
and
100
mM
2-­-mercaptoethanol
and
re-­-blotted
with
the
antibodies

GAPDH
(1:2,500)
(Abcam,
Cambridge,
UK)
and
ERK2
(1:2,000)
(Santa
Cruz

Biotechnology).
Films
were
scanned
and
a
densitometric
analysis
was
performed

using
Kodak
GL
200
Imaging
system
and
Kodak
Molecular
Imaging
software.
The

intensity
of
the
bands
quantified
by
densitometry
was
graphed.
Data
were

normalized
by
FGFR3
protein
levels
of
the
scramble
control,
defined
as
100%.
In

the
case
of
ERK1/2
phosphorylation,
data
were
normalized
by
ERK1/2

phosphorylation
levels
of
the
scramble
control,
defined
as
100%.
All
the
data

shown
are
representative
of
three
independent
experiments.

2.5.
Statistical
Analysis

The
differences
between
the
mean
values
were
analyzed
with
InStat3

software
(GraphPad
Software,
La
Jolla,
CA,
USA)
using
ANOVA
test;
statistical

significance
was
considered
to
be
achieved
at
the
P
<
0.05
level.

3. RESULTS

3.1.
Silencing
of
FGFR3
in
human
achondroplastic
chondrocytes
by
siRNAs

siRNAs
were
transfected
into
human
achondroplastic
chondrocytes
and
72

h
later
the
ability
of
the
selected
siRNA
sequences
to
knockdown
FGFR3
mRNA
and

protein
levels
was
analyzed
by
RT-­-PCR
and
Western
Blot,
respectively
(Figure
1).

Of
the
three
siRNA
sequences
tested
in
achondroplastic
chondrocytes,
sequences
1

and
2
showed
55%
and
75%
knockdown
of
FGFR3
mRNA
expression,
respectively,

as
compared
to
scramble
control
(Figure
1A).
siRNA
sequence
3
was
less
effective

at
reducing
FGFR3
mRNA
expression
and
only
38%
reduction
was
detected.

Western
blot
analysis
of
cell
culture
lysates
from
these
cells
revealed
that

the
amount
of
FGFR3
protein
also
decreased
(Figure
1B).
In
particular,

densitometry
of
immunoblots
indicated
that
FGFR3
protein
was
reduced
by
52%

and
61%
with
sequence
1
and
2,
respectively.
Once
more,
the
sequence
3
induced
a

lower
decrease
as
compared
to
the
scramble
control
(20%
of
reduction).

3.2.
Effect
of
FGFR3
knockdown
on
ERK1/2
activation

To
investigate
the
impact
of
FGFR3
silencing
on
downstream
signalling,
the

phosphorylation
status
of
ERK1/2
was
evaluated
(Figure
2).
siRNA
sequence
2

reduced
ERK1/2
phosphorylation
by
41%
as
compared
to
scramble
control
levels.

Although
to
a
lesser
extent,
siRNA
sequence
1
also
decreased
the
levels
of

phosphorylated
ERK1/2
(27%
of
reduction),
whereas
siRNA
sequence
3
hardly

modified
the
degree
of
phosphorylation
of
ERK1/2
when
compared
to
scramble

control.

7