SUPPLEMENTARY INFORMATION Supplementary Figure S1. Efficacy of STIM1 knockdown by electroporation of STIM1 sirnas. (a) Western blot of FDB muscles from 4 mice treated with either STIM1-specific sirna (KD) or negative control sirna (Ctrl). 5 μg of protein was loaded into each lane. (b) Quantification of both upper (STIM1L) and lower (STIM1S) STIM1 bands normalized to a non-specific band as loading control. Comparable knockdown was observed for both STIM1L (80±5%) and STIM1S (62±12%). *p<0.05 by Student s t-test, n=4 mice. Error bars represent s.e.m.
Supplementary Figure S2. Repetitive high frequency tetanic stimulation protocol. (a) (Top) Schematic diagram of the repetitive high frequency tetanic stimulation protocol (50 Hz, 500 ms, every 2.5 seconds, 60 tetani) used to stimulate Ca 2+ transients in single FDB fibres. (Bottom) Representative raw mag-fluo-4 fluorescence trace elicited during a single tetanus (500 ms at 50 Hz). The regions used to calculate baseline, peak, and tail integral (hatched area) are indicated. (b) Representative raw mag-fluo-4 fluorescence traces (for tetani 2, 30, 60) from a FDB fibre of a WT mouse elicited during repetitive high frequency tetanic stimulation in the absence (left) and presence of 0.2 mm La 3+ plus 0.5 mm Cd 2+ (right).
Supplementary Figure S3. Relative expression of endogenous STIM1, endogenous Orai1, and dnorai1 protein in dnorai1 mice. (a) (Left) Western blot analysis and quantification (right) of endogenous WT Orai1 and dnorai1 in TA muscles of 3 WT and 3 dnorai1 line 5 transgenic (TG) mice. 5 μg of protein was loaded into each lane and α-actinin was used as a loading control. (b) (Left) Western blot analysis and quantification (right) of endogenous STIM1S and STIM1L from TA muscles from 3 WT and 3 dnorai1 line 5 TG mice. 5 μg of protein was loaded into each lane and GAPDH was used as a loading control. Error bars represent s.e.m.
Supplementary Figure S4. Relative SERCA, DHPR 1S, CSQ1 and RyR1 expression are unaltered in dnorai1 transgenic mice. (a) (Left) Western blot analysis and quantification (right) of SERCA in TA muscles from 3 WT and 3 dnorai1 line 5 transgenic (TG) mice. (b) (Left) Western blot analysis and quantification (right) of RyR1 (upper blot) and CSQ1, (lower blot) in TA muscles from 3 WT and 3 dnorai1 line 5 TG mice. (c) (Left) Western blot analysis and quantification (right) of DHPR 1S in TA muscles from 3 WT and 3 dnorai1 line 5 TG mice. 5 μg of protein was loaded into each lane and GAPDH was used as a loading control. Error bars represent s.e.m.
WT WT dnorai1 WT dnorai1 τ decay (ms) a WT dnorai1 0.2 ΔF/F 0 b 0.5 0.5 s c 20 Peak F/F 0 0.4 0.3 0.2 0.1 15 10 5 0.0 0 d 0.1 ΔR 10 s Ionomycin/CPA/EGTA/0Ca WT dnorai1 e Fura2FF Ratio 340/380 0.5 0.4 0.3 0.2 0.1 0.0 *dnorai1 Supplementary Figure S5. Electrically-evoked Ca 2+ release and reuptake is unaltered, but SR Ca 2+ content is reduced in FDB fibres from dnorai1 transgenic mice. (a) Representative mag-fluo-4 fluorescence traces elicited by 3 consecutive electrically-evoked twitch stimuli (1Hz) in FDB fibres from WT (left) and dnorai1 line 5 TG mice (right). (b) Average (± s.e.m.) peak increase in relative mag-fluo-4 fluorescence ( F/F 0 ) amplitude during 1 Hz electrical stimulation in FDB fibres from WT (n=15) and dnorai1 line 5 TG mice (n=17). (c) Average (±s.e.m.) time constant of decay of electrically-evoked magfluo-4 twitch transients in FDB fibres from WT (n=15) and dnorai1 TG mice (n=17). (d-e) Representative fura2-ff fluorescence traces (d) and average (± s.e.m.) increase in Fura2-FF 340/380nm ratio (e) for WT and dnorai FDB fibres during application of Ca 2+ store release cocktail (10 μm ionomycin, 30 μm CPA, and 100 μm EGTA/0 Ca 2+ ). *p<0.01 by Student s-t test. n=11 for WT and n=6 for dnorai1.
Supplementary Figure S6. Reduced peak force and increased susceptibility to fatigue in EDL from dnorai1 line #86 mice. (a) Average (±s.e.m.) peak specific force during a single 500 ms tetanus at 150 Hz in EDL from WT (Black bar, n=10 muscles from 5 mice) and dnorai1 line #86 (Grey bar, n=4 muscles from 2 mice). * P<0.01 student s-t test. (b) Average (±s.e.m.) peak specific force during 60 consecutive tetanic stimuli (500 ms, at 50Hz, every 2.5 s) in EDL muscles from WT (filled circles,, n=8 muscles from 4 mice) or line 86 dnorai1 mice (open circles,, n=4 muscles from 2 mice).
Supplementary Figure S7. Full length images for immunoblots. Red boxes indicate the cropped images used in the figures. A single blot was cut and probed using different primary antibodies as indicated.
SUPPLEMENTARY TABLES Supplementary Table S1. Sequences of STIM1 sirnas and negative control sirnas. Target Sequence STIM1 Neg. Control GUGCAGUACUACAACAUCA GUGAUGAGUUCCUAAGGGA CGAAACAUCCAUAAGCUGA GCACCGAACUGUGGAAGUA UAGCGACUAAACACAUCAA UAAGGCUAUGAAGAGAUAC AUGUAUUGGCCUGUAUUAG AUGAACGUGAAUUGCUCAA
Supplementary Table S2. Primary antibodies for western blot and immunocytochemistry (ICC). Antigen Host Dilution Manufacturer Usage GOK/STIM1 HA-tag DHPR RyR1, 34C Mouse, Mouse, Mouse, Mouse, 1:350 BD Bioscience Western blot 1:10000/1:3000 Covance Western blot/icc 1:400 Thermo Fisher Western blot Scientific 1:40/1:30 Developmental Studies Western Hybridoma Bank, blot/icc University of Iowa 1:5000 Thermo Fisher Western blot Scientific Calsequestrin Mouse, SERCA Rabbit, poly 1:5000 Santa Cruz Western blot GAPDH Mouse, 1:50000 Applied Western blot Biosystems/Ambion α-actinin, EA-53 Mouse, 1:5000/1:200 Sigma-Aldrich Western blot/icc Orai1 N-terminus Rabbit, poly 1: 1000 Gift from Prof. V. Westernblot/ICC Flockerzi STIM1 (Cterminal) Rabbit, poly 1:100 Sigma-Aldrich ICC Anti-mouse IgG 1:2000 Bio-Rad Western-blot HRP Anti-rabbit IgG 1:2000 Bio-rad Western-bot HRP Anti-mouse IgG 1:1000 Invitrogen ICC Alexa Fluor 488 Anti-rabbit IgG 1:500 Jackson ICC Rhodmine ImmunoResearch