Oláh e al. Supplemen a y in o ma ion 1
SUPPLEMENTARY INFORMATION
o he manusc ip o “O e exp ession o ansien ecep o po en ial canonical ype1
(TRPC1) al e s bo h s o e ope a ed calcium en y and depola iza ion-e oked calcium
signals in C2C12 cells” by Tamás Oláh e al.
Supplemen a y Figu e 1.
Examina ion o he e ec on SOCE o he ex en o TRPC1 o e exp ession. (A)
Wes e n-blo analysis o TRPC1 in he ans ec ed clones. The 87 kDa human iso o m
was exp essed om he plasmid used o s able ans ec ion. Clone 8 was showing he
mos signi ican o e exp ession, so his clone was used in mos o he o he expe imen s
( e e ed o as TRPC1 on he o he igu es). Clone 10 and 11 we e also used in some
expe imen s. Changes in calcium concen a ion demons a ing SOCE we e eco ded
ollowing he e-es ablishing o he no mal (1.8 mM) ex e nal calcium concen a ion
([Ca
2+
]
o
) in TRPC1 o e exp essing C2C12 clones ((B) clone 11, (C) clone 10). The
in e nal calcium-s o es we e emp ied by 4 µM hapsiga gin (TG) in he absence o
[Ca
2+
]
o
. Rep esen a i e eco ds o 2 independen cul u es. (D) Maximal a e o ise and
(E) ampli ude o SOCE in myo ubes o e exp essing TRPC1 in di e en ex en s.
As e isks (*) indica e signi ican (p < 0.05) di e ence i compa ed o pa en al cells;
pa ag aph signs (§) indica e signi ican (p < 0.05) di e ence i compa ed o mock-
ans ec ed, while “and” signs (&) indica e signi ican (p < 0.05) di e ence i compa ed
o TRPC1 o e exp essing (clone 8) myo ubes. Numbe s in pa en heses indica e he
numbe o cells measu ed.
Supplemen a y Figu e 2.
Immunocy ochemical s aining o 5-day-old C2C12 myo ubes demons a ing he
p esence o p o eins associa ed wi h calcium signaling. (A) Exp ession o STIM1
p o ein in pa en al (le panel) and in TRPC1 o e exp essing ( igh panel) C2C12
myo ubes. (B) Exp ession o O ai1 p o ein in pa en al (le panel) and in TRPC1
o e exp essing ( igh panel) C2C12 myo ubes. Nuclei we e s ained wi h DAPI.
Calib a ion is he same o all images. O iginal magni ica ion was 40×.
Oláh e al. Supplemen a y in o ma ion 2
Supplemen a y Figu e 3.
Immunocy ochemical s aining o 5-day-old C2C12 myo ubes demons a ing he
agg ega ion o STIM1 p o ein in o punc a a e s o e deple ion caused by 4 µM
hapsiga gin o 30 minu es. On he igh panel he zoom was 4×, on he same isual
ield as shown in he le panel. (A) Exp ession o STIM1 p o ein in pa en al C2C12
myo ubes. (B) Exp ession o STIM1 p o ein in mock- ans ec ed C2C12 myo ubes. (C)
Exp ession o STIM1 p o ein in TRPC1 o e exp essing C2C12 myo ubes. Nuclei we e
s ained wi h DAPI. O iginal magni ica ion was 40×.
Supplemen a y Figu e 4.
E ec s o NFAT ac i i y in pa en al and TRPC1 o e exp essing myo ubes. Cells
we e ans ec ed wi h a HSP-NRE lacZ plasmid as desc ibed by Rosenbe g and co-
wo ke s [36] (a kind gi om E. Zádo ). The ans ec ed cells exp essed β-
galac osidase om he plasmid in unc ion o NFAT ac i i y. The cells we e ixed wi h
4 ºC 0.5% glu a aldehyde (dilu ed in PBS) o 10 min, and hen washed h ee imes wi h
PBS. Then 1 mg/ml X-Gal (5-b omo-4-chlo o-3-indoyl-B-D-galac oside; om
In i ogen) was dissol ed in eac ion bu e (20 mM K
4
Fe(CN)
6
, 20 mM K
3
Fe(CN)
6
, 2
mM MgCl
2
in PBS, pH 7.8) and placed on he cells o e nigh . In he dependence o β-
galac osidase ac i i y, he cells became blue. The s ained cells we e examined by a
luo escen mic oscope. 5-5 ROIs we e selec ed on each myo ube. Quan i a i e analysis
was pe o med wi h ImageJ on 15 pa en al and 9 TRPC1 o e exp essing myo ubes
(p<0.05; 57.5±1.4 AU, n=75 in pa en al and 46.1±2.6 AU, n=45 in TRPC1
o e exp essing myo ubes). (A) Pa en al myo ubes. (B) TRPC1 o e exp essing
myo ubes. O iginal magni ica ion was 10×. (C) Rep esen a i e images o di e en ia ing
C2C12 cells on di e en days o cul u ing. Pa en al, mock- ans ec ed and TRPC1
o e exp essing C2C12 myo ubes we e cul u ed unde he same condi ions. The
p oli e a ing solu ion was exchanged o di e en ia ing solu ion on he 2
nd
day o
cul u ing. No e he hinne diame e o he e minally di e en ia ed myo ubes in he
TRPC1 o e exp essing cul u es compa ed o pa en al and mock- ans ec ed cul u es.
Oláh e al. Supplemen a y in o ma ion 3
Supplemen a y Figu e 1. (Oláh e al.)
A
B C
Clone 8
(TRPC1)
monome
(~ 90 kDa)
dime
Clone 10Clone 11
Clone 11
50 nM
4 µM TG
1.8 mM [Ca
2+
]
o
100 s
Clone 10
D E
d[Ca
2+
]
i
/ d (nM/s)
0
2
4
6
8
(20) (12) (14)
(24)
*§
&
Pa en al
Mock
Clone 11
Clone 10
Clone 8 (TRPC1)
&
&
§
(7)
4 µM TG
1.8 mM [Ca
2+
]
o
∆[Ca
2+
]
i
(nM)
0
50
100
150
200
250
(14) (12) (14)
(24)
*
*
§
§
&&*
(7)
§
Supplemen a y Figu e 1. (Oláh e al.)
A
B C
Clone 8
(TRPC1)
monome
(~ 90 kDa)
dime
Clone 10Clone 11 Clone 8
(TRPC1)
monome
(~ 90 kDa)
dime
Clone 10Clone 11
Clone 11
50 nM
4 µM TG
1.8 mM [Ca
2+
]
o
100 s
50 nM
4 µM TG
1.8 mM [Ca
2+
]
o
100 s
Clone 10
D E
d[Ca
2+
]
i
/ d (nM/s)
0
2
4
6
8
(20) (12) (14)
(24)
*§
&
Pa en al
Mock
Clone 11
Clone 10
Clone 8 (TRPC1)
&
&
§
(7)
d[Ca
2+
]
i
/ d (nM/s)
0
2
4
6
8
(20) (12) (14)
(24)
*§
&
Pa en al
Mock
Clone 11
Clone 10
Clone 8 (TRPC1)
Pa en al
Mock
Clone 11
Clone 10
Clone 8 (TRPC1)
&
&
§
(7)
4 µM TG
1.8 mM [Ca
2+
]
o
4 µM TG
1.8 mM [Ca
2+
]
o
∆[Ca
2+
]
i
(nM)
0
50
100
150
200
250
(14) (12) (14)
(24)
*
*
§
§
&&*
(7)
§
∆[Ca
2+
]
i
(nM)
0
50
100
150
200
250
(14) (12) (14)
(24)
*
*
§
§
&&*
(7)
§
Oláh e al. Supplemen a y in o ma ion 4
Supplemen a y Figu e 2. (Oláh e al.)
40 µm
A
B
Supplemen a y Figu e 2. (Oláh e al.)
40 µm40 µm
A
B
Oláh e al. Supplemen a y in o ma ion 5
Supplemen a y Figu e 3. (Oláh e al.)
A
B
40 µm 10 µm
C
Supplemen a y Figu e 3. (Oláh e al.)
A
B
40 µm40 µm 10 µm10 µm
C

Oláh e al. Supplemen a y in o ma ion 6
Supplemen a y Figu e 4. (Oláh e al.)
A B
Pa en al TRPC1
Pa en al
Mock
TRPC1
Day 1. Te minally di e en ia ed myo ubes
(Day 6-7.)
C
Day 4.
Supplemen a y Figu e 4. (Oláh e al.)
A B
Pa en al TRPC1
Pa en al
Mock
TRPC1
Day 1. Te minally di e en ia ed myo ubes
(Day 6-7.)
C
Day 4.