Supplementary Materials
STIM2 drives Ca 2+ oscillations through store-operated Ca 2+ entry caused by mild store depletion
Markus Thiel 1,2 , Annette J. Lis 1,3 and Reinhold Penner 1
1
From the Center for Biomedical Research at The Queen’s Medical Center and University of
Hawaii, Honolulu, Hawaii 96813.
2
Department of Internal Medicine I,Saarland University, D-66421 Homburg, Germany
3
Institute of Biophysics, Saarland University, D-66421 Homburg, Germany
Figure S1
Supplemental Fig. 1 . Effects of STIM2 antibody on vanadate-induced I
CRAC
in RBL-2H3 cells. Left panel shows average CRAC current densities elicited by 50 µM vanadate in the absence (black, n = 8) and presence (red, n = 3) of 10 µg/ml C-terminal STIM2. Right panel shows average I/V relationships of CRAC currents recorded at 160 s from representative cells.

Thiel et al. STIM2 drives Ca 2+ oscillations through store-operated Ca 2+ entry
Figure S2
Supplemental Fig. 2 . Effects of G418 I
CRAC
elicited by passive store-depletion in RBL-2H3 cells. [Ca
2+
] i
was clamped to 150 nM.
Currents at –80 mV of individual cells were normalized to the cell size, averaged and plotted versus time. Data represent leak-subtracted current densities (pA/pF) evoked by 50 ms voltage ramps from –100 to +100 mV. Error bars indicate
SEM. A) Average CRAC current densities induced by 0 nM Ca
2+
(20 mM BAPTA) in the absence (black, n = 9) or presence (red, n = 7) of 10 µM G418; B) 2 µM thapsigargin (TG) in the absence (black, n = 6) or presence (red, n = 5) of 10 µM G418; C) 20 µM tBHQ (n = 8).
D-F) Average I/V relationships of CRAC currents recorded at 200 s from representative cells shown in (A, B, C).
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Thiel et al. STIM2 drives Ca 2+ oscillations through store-operated Ca 2+ entry
Figure S3
Supplemental Fig. 3 . G418 inhibits TG-mediated Ca
2+
signals in RBL-2H3 cells. The bar indicates the application of TG or saline. Average changes in intracellular Ca 2+ (F
340
/F
380
) in cells pre-incubated in the absence (black) or presence of G418 (red) after 2 µM (A; black, n =
142; red, n = 134), 200 nM (B; black, n = 83; red, n = 76) and 20 nM (C; black, n = 110; red, n
= 147) application of TG. (D) Percentage of STIM2 component of total Ca 2+ influx from (A-
C). Error bars indicate SEM. Average of intracellular Ca 2+ changes (F
340
/F
380
) in Ca 2+ free medium both in the absence (black) or pre-incubated for 1 h with 500 µg/ml G418 (red) and stimulated with 2 µM TG (E; black, n = 144; red, n = 89) or Ca 2+
-containg control saline (F; black, n = 144; red, n = 89).
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Thiel et al. STIM2 drives Ca 2+ oscillations through store-operated Ca 2+ entry
Figure S4
Supplemental Fig. 4.
Vanadate induced currents require D -CRACM expression in Kc cells.
Internal solution contained 300 nM [Ca 2+ ] i
. Error bars indicate SEM. A) Average CRAC current densities at –80 mV induced by 0 µM (black, n = 5), 10 µM (black open, n = 5), 30
µM (green, n = 6), 50 µM (blue, n = 5) or 500 µM (red, n = 7) vanadate. B) Average I/V relationships of D -CRACM currents extracted from representative cells shown in (A) at 200 s.
C) Average CRAC current densities with 30 µM vanadate in the absence (green, n = 5) and presence (red, n = 6) of 100 µM G418. D) Average CRAC current densities at –80 mV in control Kc cells (filled circles) induced by 500 µM vanadate (red, n = 5) or 20 µM IP
3
(black, n = 10) and D -CRACM (open circles) knock down cells induced by 500 µM vanadate (red, n
= 5) or 20 µM IP
3
(black, n = 6).
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Thiel et al. STIM2 drives Ca 2+ oscillations through store-operated Ca 2+ entry
Figure S5
Supplemental Fig. 5 . Intracellular Ca
2+
changes (F
340
/F
380
) in a single representative RBL-
2H3 cell in the absence (A) or pre-incubated for 1 h with 500 µg/ml G418 (B). C) Control measurements. Average intracellular Ca
2+
changes (F
340
/F
380
) after saline stimulation in RBL-
2H3 cells in the absence (black, n = 249) or pre-incubated with G418 (red, n = 282). The arrow indicates the application of DNP-BSA or saline.
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Thiel et al. STIM2 drives Ca 2+ oscillations through store-operated Ca 2+ entry
Figure S6
Supplemental Fig. 6 . Effects of STIM1 and STIM2 siRNA. HEK293 cells were transfected with control siRNA or siRNA directed against STIM1 or STIM2. Blots were analyzed on
Day1, Day2 and Day3 post transfection. A) Blot was probed with antibody directed against
STIM1 (upper panel) and GAPDH (lower panel). B) For three independent experiments carried out as described in (A), band intensities were quantified by densitometry and normalized against control GAPDH band intensity. C) Blot was probed with antibody directed against STIM2 (upper panel) and GAPDH (lower panel). D) For three independent experiments carried out as described in (B), band intensities were quantified by densitometry and normalized against control GAPDH band intensity. Data are shown as percent of control, mean +/- S.E.M.
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Thiel et al. STIM2 drives Ca 2+ oscillations through store-operated Ca 2+ entry
Figure S7
Supplemental Fig. 7 . Inhibition of anti-CD3-mediated Ca
2+
signals in primary human lymphocytes. Arrow represents the application of anti-CD3 or saline. Error bars indicate SEM.
Average of intracellular Ca
2+
changes (F
340
/F
380
) in PBL cells pre-incubated in the absence
(black) or presence (red) of G418 stimulated with 10 µg/ml anti-CD3 (A; black, n = 303; red, n = 217), 2 µg/ml anti-CD3 (B; black, n = 253; red, n = 224), or control saline (C; black, n =
186; red, n = 210).
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