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STIM1 long and STIM1 gate differently TRPC1 during store-operated calcium entry

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STIM1 long and STIM1 gate differently TRPC1 during store-operated calcium entry

DYRDA, Agnieszka, KONIG, Stéphane, FRIEDEN, Maud

DYRDA, Agnieszka, KONIG, Stéphane, FRIEDEN, Maud. STIM1 long and STIM1 gate

differently TRPC1 during store-operated calcium entry. Cell Calcium, 2020, vol. 86, p. 102134

DOI : 10.1016/j.ceca.2019.102134 PMID : 31838437

Available at:

http://archive-ouverte.unige.ch/unige:140151

Disclaimer: layout of this document may differ from the published version.

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SUPPLEMENTARY FIGURE LEGENDS

Figure S1. Cells displaying ITRPC1 can be subdivided into two populations.

Using ICRAC+TRPC1 amplitude at +80 mV and Erev as criteria, cells overexpressing Orai1 and either STIM1 (black curves, O1S1) or STIM1L (red curves, O1S1L) were subdivided into two groups: (A) cells displaying a small outward current at +80 mV (≤ 1 pA/pF) and Erev at ~+35mV and (B) cells displaying a large outward current at +80 mV ( > 1pA/pF) and Erev at ~+11 mV.

I/V curves are represented as mean ± SEM. “n” represents the number of recorded cells from >

5 independent transfections.

Figure S2. Efficiency of siRNA against TRPC1 at mRNA and protein level.

(Ai) siTRPC1 efficiency was assessed by Q-PCR 48h after siRNA transfection against TRPC1 (siC1). (Aii) siTRPC1 did not affect the expression level of TRPC4, Orai1 and STIM1 mRNA (4 independent transfections, bars represent mean ± SEM). (Bi) Top panel: Endogenous TRPC1 detection by Western blot showing the decrease of TRPC1 protein level 48h after transfection against TRPC1, in conditions where STIM1 and Orai1 (O1S1) or STIM1L and Orai1 (O1S1L) were overexpressed; bottom panel: the corresponding Ponceau S staining used as a loading control. (Bii) Quantification of the Western blot obtained from 3 independent transfections in duplicate. Bars represent mean ± SEM and P > 0.05 values are in brackets.

Figure S3. STIM1 and STIM1L overexpression leads to similar protein expression levels (A) Upper panel: YFP-STIM1 and YFP-STIM1L detection by Western blot after overexpression. Endogenous STIM1 is also visible. Bottom panel: the corresponding Ponceau S staining used as a loading control. (B) Quantification (mean ± SEM) of STIM1 and STIM1L expression (3 independent transfections). No significant difference was observed between STIM1 and STIM1L expression.

Figure S4. Effects of STIM1_EE and STIM1L_EE overexpression on SOCE

HEK cells were loaded with Fura-2 to monitor changes in cytosolic Ca2+ concentration, and stimulated with 1 µM Tg to elicit SOCE. SOCE amplitudes (A) and slopes (B) were calculated upon Ca2+ re-addition in Tg-treated cells after overexpression of Orai1 with either STIM1 or STIM1_EE (O1S1, O1S1_EE; black border bars) or cells after overexpression of Orai1 with

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either STIM1L or STIM1L_EE (O1S1L, O1S1L_EE; red border bars). Numbers within the bars indicate the total number of the coverslips analyzed (between 3 and 66 cells). Bars represent mean ± SEM. Panels (C) and (D) show the amplitudes and slopes of SOCE, respectively, in cells overexpressing Orai1 with STIM1 or STIM1L (O1S1, O1S1L) and with TRPC1 (O1S1+C1, O1S1L+C1). Numbers within the bars represent the total number of coverslips analyzed (between 3 and 43 cells were analyzed per coverslip). Bars are mean ± SEM.

Figure S5. Transfection of increasing amounts of STIM1 or STIM1L plasmid with Orai1 plasmid kept constant increases the STIM1/Orai1 or STIM1L/Orai1 protein expression ratio.

(A) Upper panel: representative Western blot of cells transfected with increasing amounts (1 to 6 times) of either STIM1 (S1) or STIM1L (S1L) plasmid with Orai1 plasmid level kept constant. Bottom panel: the corresponding Ponceau S staining used as a loading control. (B) Quantification of the ratios between STIM1/Orai1 (Bi) and STIM1L /Orai1 (Bii) protein expression, obtained from 3 independent transfections. Data are mean ± SEM.

Figure S6. ICRAC amplitude is differently affected by STIM1/Orai1 and STIM1L/Orai1 ratios.

ICRAC I/V curves recorded in cells transfected with different ratios (1 to 6) of STIM1/Orai1 (A) and STIM1L/Orai1 (B). Only the amplitude of ICRAC is modified by the change in STIM1/L- Orai1 ratios, the shape and Erev of the current are not affected. For clarity, error bars are omitted.

“n” represents the number of recorded cells.

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-100 -50 50

-20 -10 O1S1, n=10 O1S1L, n=9

Erev: 353 mV Erev: 336 mV

i, pA/pF

Vm, mV

A)

-100 -50 50

-50 -30 -10 10 30 O1S1, n=11 O1S1L, n=12

Erev: 143 mV Erev: 83 mV

i, pA/pF

Vm, mV

B)

Supplementary Figure 1.

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siCtr siC1 0.0

0.5 1.0

fo ld c h a n g e re la ti v e t o s iC tr **

Ai)

TRPC4

Orai1

STIM1 0.0

0.5 1.0 1.5

fo ld c h a n g e re la ti v e t o s iC tr

Aii)

Supplementary Figure 2.

Loading control (Ponceau S)

Bii)

+siCtr +siC1

+siCtr +siC1 0.0

0.5 1.0

T R P C 1 e x p re s s io n n o rm a liz e d t o s iC tr

(0.0656)

**

O1S1 O1S1L

35 48 63 75 100 135 180 kDa 245

Bi)

TRPC1

63 75 100 kDa

Loading control (Ponceau S staining)

O 1S1+ siCtr O 1S1L +siCt r O1 S1+si C1 O 1S1L +siC1

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Supplementary Figure 3.

STIM1 STIM1L 0

20 40 60

p ro te in e x p re s s io n n o rm a liz e d t o t o ta l p ro te in l o a d

A) B)

35 48 63 75 100 135180 kDa 75 100 135 kDa

Loading control (Ponceau S staining)

STIM1 YFP-STIM1L YFP-STIM1

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Supplementary Figure 4.

O1S1

O1S1_EE

O1S1L

O1S1L_EE 0

1 2

d e lt a r a ti o ( 3 4 0 /3 8 0 ) ** *

34 10 36 16

A) ***

O1S1

O1S1_EE

O1S1L

O1S1L_EE 0.00

0.05 0.10 0.15 0.20

s lo p e , ra ti o /s e c

****

*

34 10 36 16

B)

C) D)

O1S1

O1S1+C1

O1S1L

O1S1L+C1 0.0

0.1 0.2 0.3

s lo p e , ra ti o /s e c

12 17 13 16

**

(0.0507)

O1S1

O1S1+C1

O1S1L

O1S1L+C1 0

1 2

d e lt a r a ti o ( 3 4 0 /3 8 0 )

12 17 13 16

**

***

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A)

X1 X2 X3 X6 0.000

0.005 0.010 0.015 0.020

ra ti o ( S T IM 1 /O ra i1 ), A U

✱ ✱

✱ ✱

Bi)

X1 X2 X3 X6 0.00

0.02 0.04 0.06 0.08

ra ti o ( S T IM 1 L /O ra i1 ), A U

Bii)

25 35 48 63 75 100 135180

kDa Loading control (Ponceau S staining) 75

100 135 kDa

YFP-STIM1 YFP-STIM1L STIM1

S1 S1L S1 S1L S1 S1L S1 S1L

X1 X2 X3 X6

Supplementary Figure 5.

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Supplementary Figure 6.

-100 -50 50

-10 -5

X1, n=18 X3, n=9 X6, n=6

V

m

, mV

X2, n=9

i, pA/pF STIM1

A)

-100 -50 50

-10 -5

X1, n=8 X3, n=10 X6, n=2

V

m

, mV

X2, n=4

i, pA/pF STIM1L

B)

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