Overexpression of transient receptor potential canonical type 1 (TRPC1) alters both store operated calcium entry and depolarization-evoked calcium signals in C2C12 cells

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.