# sigmar1 (Alomone Labs)

Alomone Labs is a verified supplier

Alomone Labs manufactures this product

## Structured Review

Sigmar1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more

https://www.bioz.com/result/sigmar1/product/Alomone Labs

Average 94 stars, based on 1 article reviews

Price from $9.99 to $1999.99

## Images

### 1) Product Images from "Sigma non-opioid receptor 1 is a potential therapeutic target for long QT syndrome"

**Article Title: **Sigma non-opioid receptor 1 is a potential therapeutic target for long QT syndrome

**Journal: **Nature cardiovascular research

**doi: **10.1038/s44161-021-00016-2

**Figure Legend Snippet:**( a ) Schematic representation of altered molecules (red) and therapeutic strategy using SIGMAR1 agonists (blue) in Timothy syndrome (TS) cardiomyocytes. The bottom table shows the characteristics of SIGMAR1 agonists. OCD, obsessive compulsive disorder. PBA, Pseudo Bulbar Affect. SSRI, selective serotonin reuptake inhibitor. NMDAR, N -Methyl-D-aspartate receptor. ( b ) Docking model of SIGMAR1 and its agonist PRE-084. Representative traces of 0.2Hz-paced action potentials in TS cardiomyocytes without treatment or treated with SIGMAR1 agonists, PRE-084 (+PRE), fluvoxamine (+Fluvo) or dextromethorphan (+Dxm). ( d ) Action potential duration (APD90, 90% reduction from peak) analysis in TS cardiomyocytes without treatment ( n =15) or treated with PRE ( n =13), Fluvo ( n =10) and Dxm ( n =11). Other action potential parameters are shown in – . ( e ) Representative traces of 0.5Hz-paced action potentials in isogenic control (Ctrl) iPSC-derived cardiomyocytes without treatment or treated with Dxm or Fluvo. APD90 analysis in the control cells without treatment ( n =10) or treated with Dxm ( n =11) or Fluvo ( n =10). Other action potential parameters of isogenic Ctrl are shown in – . All data are mean ± s.d. The treatment of PRE, Fluvo and Dxm for the experiments was 5μM, 2hrs. One-way ANOVA with Tukey’s multiple comparisons was used for d, f. ** P <0.01, n.s., not significant. The cardiomyocytes were from at least two independent differentiations.

**Techniques Used: **Derivative Assay

**Figure Legend Snippet:**( a ) Docking models of SIGMAR1-Fluvo, -Dxm and -PRE. ( b ) PRE-084 (5μM, 2hr) reduced CDK5 kinase activity in Timothy syndrome (TS) cardiomyocytes. PHA-793887 (PHA, 5μM), a CDK5 inhibitor, was used as positive control for the assay ( n =8/group). ( c-e ) The effects of PRE (5μM, 2hr) on CDK5R1/p35 protein ( c, d, n =12 for baseline and n =9 for 2 hr after treatment) and CDK5 protein ( c, d, n =5/group) and mRNA ( e, n =4/group). ( f ) Representative epi-fluorescent and phase-contrast images of TS cardiomyocytes without treatment, with PRE-084 (+PRE, 5μM, 2hr) or PRE-084 and NE-100 treatment (+PRE & NE-100, both 5μM, 2hr) from proximity ligation assay (PLA, SIGMAR1-CDK5, red, DAPI, blue). Scale bar, 10μm. ( g ) SIGMAR1-CDK5 PLA quantification in PRE-treated ( n =16), PRE&NE-100-treated ( n =38) and non-treated TS cardiomyocytes ( n =14). ( h ) Representative traces of relative motion analysis of TS cardiomyocyte contractions before (black) and after 2-hr PRE-084 treatment (blue). The representative traces were obtained from and . ( i-j ) Relative changes of beating rate ( i ) and irregularity ( j ) ( n =11) of TS cardiomyocytes after PRE-084 treatment. ( k ) Representative traces of Ba 2+ currents in TS cardiomyocytes without treatment or treated with PRE-084 (+PRE), or fluvoxamine (+Fluvo) (each, 5μM, 2hr) and in isogenic control cardiomyocytes (Ctrl). +Dxm representative trace is shown in . ( l ) Voltage-dependent calcium channel inactivation was significantly enhanced by PRE ( n =10), Fluvo ( n =10) and Dxm ( n =16) treatment in TS cardiomyocytes compared to non-treated cells ( n =25). n.s., no significant differences between +PRE, +Fluvo, +Dxm and isogenic Ctrl groups ( n =13). ( m ) Representative traces of Ba 2+ currents in TS cardiomyocytes before treatment and acutely treated with PRE (5μM, ~5–10 mins). ( n ) Voltage-dependent calcium channel inactivation was not significantly changed by acute PRE treatment in TS cardiomyocytes ( n =10). All data are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons was used for b, g and One-way ANOVA with Sidak’s multiple comparisons was used for l. Paired two-tailed Student’s t -test was used for i, j, n, and unpaired two-tailed Student’s t -test was used for d, e. * P <0.05, ** P <0.01, *** P <0.001, n.s., not significant. Cell samples from at least two independent differentiations were used.

**Techniques Used: **Activity Assay, Positive Control, Proximity Ligation Assay, Two Tailed Test

**Figure Legend Snippet:**( a-c ) Action potential parameters, APD50 ( a ), resting potential ( b ) and peak amplitude ( c ) in Timothy syndrome (TS) cardiomyocytes without treatment ( n =15) or treated for 2 hrs with 5μM SIGMAR1 agonists, PRE-084 (+PRE, n =13), fluvoxamine (+Fluvo, n =10) or dextromethorphan (+Dxm, n =11). APD90 is shown in . ( d-f ) Action potential parameters, APD50 ( d ), resting potential ( e ) and peak amplitude ( f ) in isogenic control (Ctrl) cardiomyocytes without treatment ( n =10) and treated for 2 hrs with 5μM SIGMAR1 agonists, Fluvo ( n =10) or Dxm ( n =11). ( g ) Quantification of human SIGMAR1 transcripts (normalized to GAPDH) in Timothy syndrome ( n =9 from two independent lines) and control cardiomyocytes ( n =12 from four independent lines). ( h ) Representative immunoblots of human SIGMAR1 and GAPDH protein using lysates from TS and control iPSC-derived cardiomyocytes. ( i-j ) Quantification of SIGMAR1 25kDa protein band ( i ) and 35kDa protein expression ( j , normalized to GAPDH) in TS iPSC-derived cardiomyocytes compared to the isogenic Ctrl ( n =6/group). The molecular weight of SIGMAR1 is ~25 kDa while the 35kDa band ( # ) has been reported previously and might be a dimer of a full-length SIGMAR1 with a SIGMAR1 splice variant . ( k ) Representative immunoblots of human ATF4 using the same lysates from TS and isogenic Ctrl iPSC-derived cardiomyocytes shown in . ( l-n ) Quantification of ATF4 38kDa protein band ( l , non-modified), 50kDa ( m , phosphorylated) and 70kDa protein expression ( n , ubiquitinated, normalized to GAPDH) in TS iPSC-derived cardiomyocytes compared to the isogenic Ctrl ( n =6/group). ( o-r ) ATF4 overexpression ( o ) significantly increased SIGMAR1 transcription ( p ) and protein expression ( q,r ) in normal human cardiomyocytes transfected with ATF4 plasmid (+ ATF4, o&p, n =7, r, n =5). The cardiomyocytes were harvested 24 hr after the lipofection. The empty vector was used as a negative control (Mock, o&p, n =5, r, n =4). ( q ) Representative immunoblots of human SIGMAR1 and GAPDH using the lysate from the transfected cardiomyocytes. All data are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons was used for a-f and unpaired two-tailed Student’s t -test was used for g, i, j, l, m, n, o, p, r. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, n.s., not significant. Cell samples from at least two independent differentiations were used.

**Techniques Used: **Western Blot, Derivative Assay, Expressing, Molecular Weight, Variant Assay, Modification, Over Expression, Transfection, Plasmid Preparation, Negative Control, Two Tailed Test

**Figure Legend Snippet:**( a-b ) Representative traces of time-course calcium imaging in spontaneously contracting isogenic control (Ctrl, black) and Timothy syndrome (TS, red) cardiomyocytes. ( c-d ) Calcium transient frequency ( c ) and duration ( d ) analysis in TS cardiomyocytes during the 2-hr imaging ( n =32). ( e ) Current-voltage relationship of Ba 2+ recordings in Dxm-treated ( n =12) and non-treated TS cardiomyocytes ( n =13). ( f ) Representative traces of Ba 2+ currents in isogenic control cardiomyocytes without treatment (Ctrl) or treated with dextromethorphan (+Dxm). ( g ) Voltage-dependent calcium channel inactivation was not altered by Dxm ( n =10) in the isogenic control cardiomyocytes compared to non-treated cells ( n =13). ( h ) Representative epi-fluorescent and phase-contrast images of TS cardiomyocytes without and with Dxm (+Dxm) from proximity ligation assay (PLA, SIGMAR1-Ca V 1.2, red, DAPI, blue). Scale bar, 10μm. ( i ) SIGMAR1-Ca V 1.2 PLA quantification in Dxm-treated ( n =33) and non-treated TS cardiomyocytes (TS, n =32). ( j ) I Kr current steady-state amplitudes were significantly reduced in TS cardiomyocytes ( n =10) compared to isogenic control ( n =10) at −10, 0 and 10 mV steps from −40mV hold. ( k ) I Kr tail currents were significantly reduced in TS cardiomyocytes ( n =10) compared to isogenic control ( n =10) at −10, 0, 10 and 20 mV steps. ( l ) Representative traces of I Kr currents (E-4031-sensitive) in isogenic Ctrl cardiomyocytes (black) and TS cardiomyocytes treated with Dxm (purple), Dxm & NE-100 (green) or without treatment (red, TS). The I Kr traces are shown in . ( m ) There was no significant difference in I Kr current (tail) between Dxm-treated ( n =9) and non-treated TS cardiomyocytes ( n =10) while Dxm & NE-100 ( n =10) significantly reduced tail currents compared to Dxm at 10, 20, 30, 40 and 50 mV steps and also to non-treated TS at 20mV step from −40mV hold. All data are mean ± s.d. The treatment of Dxm or NE-100 for all experiments was 5μM, 2hrs. One-way ANOVA with Tukey’s multiple comparisons was used for c, d between the time points to before and used for m at each voltage step. Unpaired two-tailed Student’s t -test were used for g, i between the groups and used for e, j, k at each voltage step. * P <0.05, n.s., no significant. The samples were from at least two independent differentiations.

**Techniques Used: **Imaging, Proximity Ligation Assay, Two Tailed Test

**Figure Legend Snippet:**( a ) Representative traces of time-course calcium imaging in spontaneously contracting Timothy syndrome (TS) cardiomyocytes treated with Dxm (5μM, until 120min). ( b-c ) Calcium transient frequency ( b ) and duration( c ) analysis in TS cardiomyocytes before and after Dxm treatment ( n =19). ( d ) Representative traces of Ca 2+ currents in TS cardiomyocytes with and without Dxm. ( e ) Late calcium current analysis in TS cardiomyocytes with and without Dxm (TS, n =11; +Dxm, n =12). ( f ) Representative traces of Ba 2+ currents in TS cardiomyocytes without treatment, with Dxm (5μM, 2hrs, +Dxm) or Dxm with a SIGMAR1 antagonist, NE-100 (1μM, +Dxm&NE-100). ( g ) Voltage-dependent inactivation in TS cardiomyocytes without treatment ( n =25), with Dxm ( n =16) or Dxm+NE-100 ( n =11). ( h ) Representative traces of I Kr currents (E-4031-sensitive) in TS cardiomyocytes with Dxm (5μM), Dxm&NE-100 (each, 5μM) or without treatment. ( i ) I Kr current amplitude analysis in TS cardiomyocytes with Dxm ( n =9), Dxm&NE-100 ( n =10) or without treatment ( n =10) (* P <0.05, Dxm vs TS and Dxm vs Dxm & NE-100, at −10, 0, 10, 20 and 30mV). ( j ) Representative confocal fluorescent and bright-field images of TS cardiomyocytes without and with Dxm treatment (+Dxm) from proximity ligation assay (PLA, SIGMAR1-hERG, red, DAPI, blue). Scale bar, 10μm. ( k ) SIGMAR1-hERG PLA quantification in Dxm-treated ( n =36) and non-treated ( n =35) TS cardiomyocytes. ( l ) Representative traces of I Ks currents (Chromanol-293B-sensitive) in TS cardiomyocytes treated with Dxm (5μM), Dxm&NE-100 (each, 5μM) or without treatment. ( m ) I Ks current amplitude analysis in TS cardiomyocytes with Dxm ( n =10), Dxm&NE-100 ( n =9) or without treatment ( n =10) (* P <0.05, Dxm vs TS and Dxm vs Dxm & NE-100, at −10, 0, 10, 20, 30 and 40mV). ( n ) Representative epi-fluorescent and phase-contrast images of TS cardiomyocytes without and with Dxm treatment (+Dxm) from PLA (SIGMAR1-K V 7.1, red, DAPI, blue). Scale bar, 10μm. ( o ) SIGMAR1-K V 7.1 PLA quantification in Dxm-treated ( n =40) and non-treated ( n =20) TS cardiomyocytes. All data are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons was used for b, c, g between the groups and for i, m at each voltage step. Unpaired two-tailed Student’s t -test was used for e, k, o. * P <0.05, ** P <0.01, *** P <0.001, n.s., not significant. The samples were from at least two independent differentiations.

**Techniques Used: **Imaging, Proximity Ligation Assay, Two Tailed Test

**Figure Legend Snippet:**( a ) Representative traces of I Kr current (E-4031-sensitive) in isogenic control (Ctrl) cardiomyocytes treated with Dxm (5μM, 2hr) or without treatment. ( b-c ) I Kr current amplitude ( b ) and tail I Kr current ( c ) were not significantly changed by Dxm in the isogenic Ctrl cardiomyocytes ( n =10/group). ( d ) Schematic representation of confocal imaging for proximity ligation assay (PLA, SIGMAR1-hERG, red) in human iPSC-derived cardiomyocytes. ( e ) Representative confocal fluorescent images of TS cardiomyocytes used for PLA (SIGMAR1-hERG, red) and DAPI (blue) staining. Dxm treatment (+Dxm, 5μM, 2hr) was conducted to examine the effect of Dxm on PLA signals in TS cardiomyocytes. #3 images are used in . Scale bar, 10μm. ( f ) Representative epi-fluorescent and phase-contrast images of isogenic Ctrl cardiomyocytes used for PLA (red, SIGMAR1-hERG) and DAPI (blue) staining. Dxm treatment (+Dxm, 5μM, 2hr) was conducted to examine the effect of Dxm on PLA signals in the cardiomyocytes. Scale bar, 10μm. ( g ) Quantification of PLA signal number of SIGMAR1-hERG in Dxm-treated ( n =21) and non-treated ( n =21) isogenic Ctrl cardiomyocytes. ( h ) Representative traces of I Ks current (Chromanol 293B-sensitive) in isogenic control (Ctrl) cardiomyocytes treated with Dxm or without treatment. ( i ) I Ks current amplitude analysis in the isogenic Ctrl cardiomyocytes with Dxm (5μM, 2hr, n =9) or without treatment ( n =10). ( j ) Representative epi-fluorescent and phase-contrast images of isogenic Ctrl cardiomyocytes used for PLA (red, SIGMAR1-K V 7.1) and DAPI (blue) staining. Dxm treatment (+Dxm, 5μM, 2hr) was conducted to examine the effect of Dxm on PLA signals in the cardiomyocytes. Scale bar, 10μm. ( k ) Quantification of PLA signal number of SIGMAR1-K V 7.1 in Dxm-treated ( n =40) and non-treated ( n =44) isogenic Ctrl cardiomyocytes. All data are mean ± s.d. Unpaired two-tailed Student t -test was used for g,k between the groups, and for b,c,i at each voltage step. * P <0.05. n.s., not significant. The cell samples were from at least two independent differentiations.

**Techniques Used: **Imaging, Proximity Ligation Assay, Derivative Assay, Staining, Two Tailed Test

**Figure Legend Snippet:**( a ) Representative traces of action potentials in long QT syndrome type 1 (LQTS1) iPSC-derived cardiomyocytes without treatment or treated with fluvoxamine (+Fluvo) or dextromethorphan (+Dxm). ( b ) Action potential duration (APD90) analysis in LQTS1 cardiomyocytes without treatment ( n =11) or treated with fluvoxamine ( n =10) or dextromethorphan ( n =10). Other action potential parameters are shown in , , . Representative traces of I Ks currents (Chromanol-293B-sensitive) in LQTS1 cardiomyocytes treated with Dxm or without treatment. ( d ) I Ks currents (steady-state) were significantly increased by Dxm in LQTS1 cardiomyocytes at all voltage steps ( n =10/group). Representative traces of I Kr currents (E-4031-sensitive) in LQTS1 cardiomyocytes treated with Dxm or without treatment. ( f ) I Kr currents (steady-state) were significantly increased in Dxm-treated LQTS1 cardiomyocytes ( n =9) at 0, 10, 20, 30, 40 and 50mV steps compared with LQTS1 cardiomyocytes without treatment ( n =10). ( g ) Representative epi-fluorescent and phase-contrast images of LQTS1 cardiomyocytes with and without Dxm treatment from proximity ligation assay (PLA, SIGMAR1-hERG, red, DAPI, blue). Scale bar, 10μm. ( h ) SIGMAR1-hERG PLA quantification in Dxm-treated ( n =20) and non-treated ( n =20) LQTS1 cardiomyocytes. ( i ) Representative epi-fluorescent and phase-contrast images of LQTS1 cardiomyocytes with and without Dxm treatment from PLA (SIGMAR1-K V 7.1, red, DAPI, blue). Scale bar, 10μm. ( j ) SIGMAR1-K V 7.1 PLA quantification in Dxm-treated ( n =13) and non-treated ( n =23) LQTS1 cardiomyocytes. All data are mean ± s.d. The treatment of Fluvo or Dxm for the experiments was 5μM, 2hrs. One-way ANOVA with Tukey’s multiple comparisons was used for b, and unpaired two-tailed Student’s t -test was used for h, j between the groups and for d, f at each voltage step. * P <0.05, ** P <0.01, *** P <0.001, n.s., not significant. The cardiomyocytes were from at least two independent differentiations.

**Techniques Used: **Derivative Assay, Proximity Ligation Assay, Two Tailed Test

**Figure Legend Snippet:**( a ) Representative traces of action potentials in long QT syndrome type 2 (LQTS2) iPSC-derived cardiomyocytes without treatment or treated with fluvoxamine (+Fluvo) or dextromethorphan (+Dxm). ( b ) Action potential duration (APD90) analysis in LQTS2 cardiomyocytes without treatment ( n =11) or treated with fluvoxamine ( n =10) or dextromethorphan ( n =11). Other action potential parameters are shown in , , . ( c ) Representative traces of I Kr currents (E-4031-sensitive) in LQTS2 cardiomyocytes treated with Dxm or without treatment. I Kr currents (steady-state) were significantly increased in Dxm-treated LQTS2 cardiomyocytes ( n =9) at −10 and 0mV steps compared with LQTS2 cardiomyocytes without treatment ( n =10). ( e ) Representative traces of I Ks currents (Chromanol-293B-sensitive) in LQTS2 cardiomyocytes treated with Dxm or without treatment. I Ks currents (steady-state) were significantly increased in Dxm-treated LQTS2 cardiomyocytes at 0, 10, 20, 30 and 50mV steps compared with LQTS2 cardiomyocytes without treatment ( n =10/group). ( g ) Representative epi-fluorescent and phase-contrast images of LQTS2 cardiomyocytes with and without Dxm treatment from proximity ligation assay (PLA, SIGMAR1-hERG, red, DAPI, blue). Scale bar, 10μm. ( h ) SIGMAR1-hERG PLA quantification in Dxm-treated ( n =22) and non-treated ( n =29) LQTS2 cardiomyocytes. ( i ) Representative epi-fluorescent and phase-contrast images of LQTS2 cardiomyocytes with and without Dxm treatment from PLA (SIGMAR1-K V 7.1, red, DAPI, blue). Scale bar, 10μm. ( j ) SIGMAR1-K V 7.1 PLA quantification in Dxm-treated ( n =107) and non-treated ( n =87) LQTS2 cardiomyocytes. All data are mean ± s.d. The treatment of Fluvo or Dxm for the experiments was 5μM, 2hrs. One-way ANOVA with Tukey’s multiple comparisons was used for b. Unpaired two-tailed Student’s t -test was used for h, j between the groups and for d, f at each voltage step. * P <0.05, ** P <0.01, *** P <0.001, n.s., not significant. The cardiomyocytes were from at least two independent differentiations.

**Techniques Used: **Derivative Assay, Proximity Ligation Assay, Two Tailed Test

**Figure Legend Snippet:**( a-f ) Analysis of APD50 (50% from peak, a, b ), peak amplitude ( c, d ) and resting potential ( e, f ) in LQTS1 cardiomyocytes without treatment ( n =11) or treated with fluvoxamine (Fluvo, n =10) or dextromethorphan (Dxm, n =10) and in LQTS2 cardiomyocytes without treatment ( n =11) or treated with Fluvo ( n =10) or Dxm ( n =11). ( g,h ) I Kr current (tail) was significantly increased by Dxm in LQTS1 cardiomyocytes at 40mV step ( g, LQTS1, n =10; +Dxm, n =9) and in LQTS2 cardiomyocytes at 10, 30 and 40mV steps ( h, LQTS2, n =10; +Dxm, n =9). ( i,j ) Representative traces of Ba 2+ currents in LQTS1 ( i ) or LQTS2 cardiomyocytes( j ) without treatment or treated with Dxm. ( k,l ) Voltage-dependent calcium channel inactivation was not significantly changed by Dxm in LQTS1( k ) or LQTS2 cardiomyocytes ( l ) compared to non-treated cells ( n =10/group). ( m,n ) Current-voltage relationship of Ba 2+ recordings in Dxm-treated ( n =10) and non-treated LQTS1 cardiomyocytes ( n =7)( m ) and in Dxm-treated ( n =7) and non-treated LQTS2 cardiomyocytes ( n =8)( n ). ( o,p ) Representative immunoblots of human SIGMAR1, ATF4, CDK5, CDK5R1/p35 and GAPDH protein using lysates from control (Ctrl) cardiomyocytes, LQTS1 cardiomyocytes ( o ) and LQTS2 cardiomyocytes ( p ). ^, p35 antibody used for was discontinued (Santa Cruz, sc-820). Therefore, another antibody (Cell Signaling Technology, C64B10) was used for this blotting series while its signal-to-noise ratio was not as high as sc-820. ( q-s ) Quantification of SIGMAR1 25kDa protein band ( q, r ) and # 35kDa protein band ( s , possibly a dimer of a full-length SIGMAR1 with a SIGMAR1 splice variant ) in LQTS1 or LQTS2 cardiomyocytes compared to Ctrl ( n =4/group). ( t,u ) Quantification of CDK5 (left) and CDK5R1/p35 protein expressions (right) in LQTS1 or LQTS2 cardiomyocytes compared to control cardiomyocytes ( n =4/group). ( v,w ) Quantification of ATF4 38kDa protein band (left, non-modified), 50kDa (center, phosphorylated) and 70kDa protein expression (right, ubiquitinated) in LQTS1 or LQTS2 cardiomyocytes compared to control cardiomyocytes ( n =4/group). All data are mean ± s.d. The treatment of Dxm or Fluvo for the experiments was 5μM, 2hrs. One-way ANOVA with Tukey’s multiple comparisons was used for a,b,c,d,e,f. Unpaired two-tailed Student’s t -test was used for k,l,q,r,s,t,u,v,w between the groups and for g,h,m,n at each voltage step. * P <0.05, ** P <0.01, n.s., not significant. The cardiomyocytes were from at least two independent differentiations.

**Techniques Used: **Western Blot, Variant Assay, Modification, Expressing, Two Tailed Test

# sigmar1 (Alomone Labs)

Alomone Labs is a verified supplier

Alomone Labs manufactures this product

## Structured Review

Sigmar1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more

https://www.bioz.com/result/sigmar1/product/Alomone Labs

Average 94 stars, based on 1 article reviews

Price from $9.99 to $1999.99

## Images

### 1) Product Images from "Sigma non-opioid receptor 1 is a potential therapeutic target for long QT syndrome"

**Article Title: **Sigma non-opioid receptor 1 is a potential therapeutic target for long QT syndrome

**Journal: **Nature cardiovascular research

**doi: **10.1038/s44161-021-00016-2

**Figure Legend Snippet:**( a ) Schematic representation of altered molecules (red) and therapeutic strategy using SIGMAR1 agonists (blue) in Timothy syndrome (TS) cardiomyocytes. The bottom table shows the characteristics of SIGMAR1 agonists. OCD, obsessive compulsive disorder. PBA, Pseudo Bulbar Affect. SSRI, selective serotonin reuptake inhibitor. NMDAR, N -Methyl-D-aspartate receptor. ( b ) Docking model of SIGMAR1 and its agonist PRE-084. Representative traces of 0.2Hz-paced action potentials in TS cardiomyocytes without treatment or treated with SIGMAR1 agonists, PRE-084 (+PRE), fluvoxamine (+Fluvo) or dextromethorphan (+Dxm). ( d ) Action potential duration (APD90, 90% reduction from peak) analysis in TS cardiomyocytes without treatment ( n =15) or treated with PRE ( n =13), Fluvo ( n =10) and Dxm ( n =11). Other action potential parameters are shown in – . ( e ) Representative traces of 0.5Hz-paced action potentials in isogenic control (Ctrl) iPSC-derived cardiomyocytes without treatment or treated with Dxm or Fluvo. APD90 analysis in the control cells without treatment ( n =10) or treated with Dxm ( n =11) or Fluvo ( n =10). Other action potential parameters of isogenic Ctrl are shown in – . All data are mean ± s.d. The treatment of PRE, Fluvo and Dxm for the experiments was 5μM, 2hrs. One-way ANOVA with Tukey’s multiple comparisons was used for d, f. ** P <0.01, n.s., not significant. The cardiomyocytes were from at least two independent differentiations.

**Techniques Used: **Derivative Assay

**Figure Legend Snippet:**( a ) Docking models of SIGMAR1-Fluvo, -Dxm and -PRE. ( b ) PRE-084 (5μM, 2hr) reduced CDK5 kinase activity in Timothy syndrome (TS) cardiomyocytes. PHA-793887 (PHA, 5μM), a CDK5 inhibitor, was used as positive control for the assay ( n =8/group). ( c-e ) The effects of PRE (5μM, 2hr) on CDK5R1/p35 protein ( c, d, n =12 for baseline and n =9 for 2 hr after treatment) and CDK5 protein ( c, d, n =5/group) and mRNA ( e, n =4/group). ( f ) Representative epi-fluorescent and phase-contrast images of TS cardiomyocytes without treatment, with PRE-084 (+PRE, 5μM, 2hr) or PRE-084 and NE-100 treatment (+PRE & NE-100, both 5μM, 2hr) from proximity ligation assay (PLA, SIGMAR1-CDK5, red, DAPI, blue). Scale bar, 10μm. ( g ) SIGMAR1-CDK5 PLA quantification in PRE-treated ( n =16), PRE&NE-100-treated ( n =38) and non-treated TS cardiomyocytes ( n =14). ( h ) Representative traces of relative motion analysis of TS cardiomyocyte contractions before (black) and after 2-hr PRE-084 treatment (blue). The representative traces were obtained from and . ( i-j ) Relative changes of beating rate ( i ) and irregularity ( j ) ( n =11) of TS cardiomyocytes after PRE-084 treatment. ( k ) Representative traces of Ba 2+ currents in TS cardiomyocytes without treatment or treated with PRE-084 (+PRE), or fluvoxamine (+Fluvo) (each, 5μM, 2hr) and in isogenic control cardiomyocytes (Ctrl). +Dxm representative trace is shown in . ( l ) Voltage-dependent calcium channel inactivation was significantly enhanced by PRE ( n =10), Fluvo ( n =10) and Dxm ( n =16) treatment in TS cardiomyocytes compared to non-treated cells ( n =25). n.s., no significant differences between +PRE, +Fluvo, +Dxm and isogenic Ctrl groups ( n =13). ( m ) Representative traces of Ba 2+ currents in TS cardiomyocytes before treatment and acutely treated with PRE (5μM, ~5–10 mins). ( n ) Voltage-dependent calcium channel inactivation was not significantly changed by acute PRE treatment in TS cardiomyocytes ( n =10). All data are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons was used for b, g and One-way ANOVA with Sidak’s multiple comparisons was used for l. Paired two-tailed Student’s t -test was used for i, j, n, and unpaired two-tailed Student’s t -test was used for d, e. * P <0.05, ** P <0.01, *** P <0.001, n.s., not significant. Cell samples from at least two independent differentiations were used.

**Techniques Used: **Activity Assay, Positive Control, Proximity Ligation Assay, Two Tailed Test

**Figure Legend Snippet:**( a-c ) Action potential parameters, APD50 ( a ), resting potential ( b ) and peak amplitude ( c ) in Timothy syndrome (TS) cardiomyocytes without treatment ( n =15) or treated for 2 hrs with 5μM SIGMAR1 agonists, PRE-084 (+PRE, n =13), fluvoxamine (+Fluvo, n =10) or dextromethorphan (+Dxm, n =11). APD90 is shown in . ( d-f ) Action potential parameters, APD50 ( d ), resting potential ( e ) and peak amplitude ( f ) in isogenic control (Ctrl) cardiomyocytes without treatment ( n =10) and treated for 2 hrs with 5μM SIGMAR1 agonists, Fluvo ( n =10) or Dxm ( n =11). ( g ) Quantification of human SIGMAR1 transcripts (normalized to GAPDH) in Timothy syndrome ( n =9 from two independent lines) and control cardiomyocytes ( n =12 from four independent lines). ( h ) Representative immunoblots of human SIGMAR1 and GAPDH protein using lysates from TS and control iPSC-derived cardiomyocytes. ( i-j ) Quantification of SIGMAR1 25kDa protein band ( i ) and 35kDa protein expression ( j , normalized to GAPDH) in TS iPSC-derived cardiomyocytes compared to the isogenic Ctrl ( n =6/group). The molecular weight of SIGMAR1 is ~25 kDa while the 35kDa band ( # ) has been reported previously and might be a dimer of a full-length SIGMAR1 with a SIGMAR1 splice variant . ( k ) Representative immunoblots of human ATF4 using the same lysates from TS and isogenic Ctrl iPSC-derived cardiomyocytes shown in . ( l-n ) Quantification of ATF4 38kDa protein band ( l , non-modified), 50kDa ( m , phosphorylated) and 70kDa protein expression ( n , ubiquitinated, normalized to GAPDH) in TS iPSC-derived cardiomyocytes compared to the isogenic Ctrl ( n =6/group). ( o-r ) ATF4 overexpression ( o ) significantly increased SIGMAR1 transcription ( p ) and protein expression ( q,r ) in normal human cardiomyocytes transfected with ATF4 plasmid (+ ATF4, o&p, n =7, r, n =5). The cardiomyocytes were harvested 24 hr after the lipofection. The empty vector was used as a negative control (Mock, o&p, n =5, r, n =4). ( q ) Representative immunoblots of human SIGMAR1 and GAPDH using the lysate from the transfected cardiomyocytes. All data are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons was used for a-f and unpaired two-tailed Student’s t -test was used for g, i, j, l, m, n, o, p, r. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, n.s., not significant. Cell samples from at least two independent differentiations were used.

**Techniques Used: **Western Blot, Derivative Assay, Expressing, Molecular Weight, Variant Assay, Modification, Over Expression, Transfection, Plasmid Preparation, Negative Control, Two Tailed Test

**Figure Legend Snippet:**( a-b ) Representative traces of time-course calcium imaging in spontaneously contracting isogenic control (Ctrl, black) and Timothy syndrome (TS, red) cardiomyocytes. ( c-d ) Calcium transient frequency ( c ) and duration ( d ) analysis in TS cardiomyocytes during the 2-hr imaging ( n =32). ( e ) Current-voltage relationship of Ba 2+ recordings in Dxm-treated ( n =12) and non-treated TS cardiomyocytes ( n =13). ( f ) Representative traces of Ba 2+ currents in isogenic control cardiomyocytes without treatment (Ctrl) or treated with dextromethorphan (+Dxm). ( g ) Voltage-dependent calcium channel inactivation was not altered by Dxm ( n =10) in the isogenic control cardiomyocytes compared to non-treated cells ( n =13). ( h ) Representative epi-fluorescent and phase-contrast images of TS cardiomyocytes without and with Dxm (+Dxm) from proximity ligation assay (PLA, SIGMAR1-Ca V 1.2, red, DAPI, blue). Scale bar, 10μm. ( i ) SIGMAR1-Ca V 1.2 PLA quantification in Dxm-treated ( n =33) and non-treated TS cardiomyocytes (TS, n =32). ( j ) I Kr current steady-state amplitudes were significantly reduced in TS cardiomyocytes ( n =10) compared to isogenic control ( n =10) at −10, 0 and 10 mV steps from −40mV hold. ( k ) I Kr tail currents were significantly reduced in TS cardiomyocytes ( n =10) compared to isogenic control ( n =10) at −10, 0, 10 and 20 mV steps. ( l ) Representative traces of I Kr currents (E-4031-sensitive) in isogenic Ctrl cardiomyocytes (black) and TS cardiomyocytes treated with Dxm (purple), Dxm & NE-100 (green) or without treatment (red, TS). The I Kr traces are shown in . ( m ) There was no significant difference in I Kr current (tail) between Dxm-treated ( n =9) and non-treated TS cardiomyocytes ( n =10) while Dxm & NE-100 ( n =10) significantly reduced tail currents compared to Dxm at 10, 20, 30, 40 and 50 mV steps and also to non-treated TS at 20mV step from −40mV hold. All data are mean ± s.d. The treatment of Dxm or NE-100 for all experiments was 5μM, 2hrs. One-way ANOVA with Tukey’s multiple comparisons was used for c, d between the time points to before and used for m at each voltage step. Unpaired two-tailed Student’s t -test were used for g, i between the groups and used for e, j, k at each voltage step. * P <0.05, n.s., no significant. The samples were from at least two independent differentiations.

**Techniques Used: **Imaging, Proximity Ligation Assay, Two Tailed Test

**Figure Legend Snippet:**( a ) Representative traces of time-course calcium imaging in spontaneously contracting Timothy syndrome (TS) cardiomyocytes treated with Dxm (5μM, until 120min). ( b-c ) Calcium transient frequency ( b ) and duration( c ) analysis in TS cardiomyocytes before and after Dxm treatment ( n =19). ( d ) Representative traces of Ca 2+ currents in TS cardiomyocytes with and without Dxm. ( e ) Late calcium current analysis in TS cardiomyocytes with and without Dxm (TS, n =11; +Dxm, n =12). ( f ) Representative traces of Ba 2+ currents in TS cardiomyocytes without treatment, with Dxm (5μM, 2hrs, +Dxm) or Dxm with a SIGMAR1 antagonist, NE-100 (1μM, +Dxm&NE-100). ( g ) Voltage-dependent inactivation in TS cardiomyocytes without treatment ( n =25), with Dxm ( n =16) or Dxm+NE-100 ( n =11). ( h ) Representative traces of I Kr currents (E-4031-sensitive) in TS cardiomyocytes with Dxm (5μM), Dxm&NE-100 (each, 5μM) or without treatment. ( i ) I Kr current amplitude analysis in TS cardiomyocytes with Dxm ( n =9), Dxm&NE-100 ( n =10) or without treatment ( n =10) (* P <0.05, Dxm vs TS and Dxm vs Dxm & NE-100, at −10, 0, 10, 20 and 30mV). ( j ) Representative confocal fluorescent and bright-field images of TS cardiomyocytes without and with Dxm treatment (+Dxm) from proximity ligation assay (PLA, SIGMAR1-hERG, red, DAPI, blue). Scale bar, 10μm. ( k ) SIGMAR1-hERG PLA quantification in Dxm-treated ( n =36) and non-treated ( n =35) TS cardiomyocytes. ( l ) Representative traces of I Ks currents (Chromanol-293B-sensitive) in TS cardiomyocytes treated with Dxm (5μM), Dxm&NE-100 (each, 5μM) or without treatment. ( m ) I Ks current amplitude analysis in TS cardiomyocytes with Dxm ( n =10), Dxm&NE-100 ( n =9) or without treatment ( n =10) (* P <0.05, Dxm vs TS and Dxm vs Dxm & NE-100, at −10, 0, 10, 20, 30 and 40mV). ( n ) Representative epi-fluorescent and phase-contrast images of TS cardiomyocytes without and with Dxm treatment (+Dxm) from PLA (SIGMAR1-K V 7.1, red, DAPI, blue). Scale bar, 10μm. ( o ) SIGMAR1-K V 7.1 PLA quantification in Dxm-treated ( n =40) and non-treated ( n =20) TS cardiomyocytes. All data are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons was used for b, c, g between the groups and for i, m at each voltage step. Unpaired two-tailed Student’s t -test was used for e, k, o. * P <0.05, ** P <0.01, *** P <0.001, n.s., not significant. The samples were from at least two independent differentiations.

**Techniques Used: **Imaging, Proximity Ligation Assay, Two Tailed Test

**Figure Legend Snippet:**( a ) Representative traces of I Kr current (E-4031-sensitive) in isogenic control (Ctrl) cardiomyocytes treated with Dxm (5μM, 2hr) or without treatment. ( b-c ) I Kr current amplitude ( b ) and tail I Kr current ( c ) were not significantly changed by Dxm in the isogenic Ctrl cardiomyocytes ( n =10/group). ( d ) Schematic representation of confocal imaging for proximity ligation assay (PLA, SIGMAR1-hERG, red) in human iPSC-derived cardiomyocytes. ( e ) Representative confocal fluorescent images of TS cardiomyocytes used for PLA (SIGMAR1-hERG, red) and DAPI (blue) staining. Dxm treatment (+Dxm, 5μM, 2hr) was conducted to examine the effect of Dxm on PLA signals in TS cardiomyocytes. #3 images are used in . Scale bar, 10μm. ( f ) Representative epi-fluorescent and phase-contrast images of isogenic Ctrl cardiomyocytes used for PLA (red, SIGMAR1-hERG) and DAPI (blue) staining. Dxm treatment (+Dxm, 5μM, 2hr) was conducted to examine the effect of Dxm on PLA signals in the cardiomyocytes. Scale bar, 10μm. ( g ) Quantification of PLA signal number of SIGMAR1-hERG in Dxm-treated ( n =21) and non-treated ( n =21) isogenic Ctrl cardiomyocytes. ( h ) Representative traces of I Ks current (Chromanol 293B-sensitive) in isogenic control (Ctrl) cardiomyocytes treated with Dxm or without treatment. ( i ) I Ks current amplitude analysis in the isogenic Ctrl cardiomyocytes with Dxm (5μM, 2hr, n =9) or without treatment ( n =10). ( j ) Representative epi-fluorescent and phase-contrast images of isogenic Ctrl cardiomyocytes used for PLA (red, SIGMAR1-K V 7.1) and DAPI (blue) staining. Dxm treatment (+Dxm, 5μM, 2hr) was conducted to examine the effect of Dxm on PLA signals in the cardiomyocytes. Scale bar, 10μm. ( k ) Quantification of PLA signal number of SIGMAR1-K V 7.1 in Dxm-treated ( n =40) and non-treated ( n =44) isogenic Ctrl cardiomyocytes. All data are mean ± s.d. Unpaired two-tailed Student t -test was used for g,k between the groups, and for b,c,i at each voltage step. * P <0.05. n.s., not significant. The cell samples were from at least two independent differentiations.

**Techniques Used: **Imaging, Proximity Ligation Assay, Derivative Assay, Staining, Two Tailed Test

**Figure Legend Snippet:**( a ) Representative traces of action potentials in long QT syndrome type 1 (LQTS1) iPSC-derived cardiomyocytes without treatment or treated with fluvoxamine (+Fluvo) or dextromethorphan (+Dxm). ( b ) Action potential duration (APD90) analysis in LQTS1 cardiomyocytes without treatment ( n =11) or treated with fluvoxamine ( n =10) or dextromethorphan ( n =10). Other action potential parameters are shown in , , . Representative traces of I Ks currents (Chromanol-293B-sensitive) in LQTS1 cardiomyocytes treated with Dxm or without treatment. ( d ) I Ks currents (steady-state) were significantly increased by Dxm in LQTS1 cardiomyocytes at all voltage steps ( n =10/group). Representative traces of I Kr currents (E-4031-sensitive) in LQTS1 cardiomyocytes treated with Dxm or without treatment. ( f ) I Kr currents (steady-state) were significantly increased in Dxm-treated LQTS1 cardiomyocytes ( n =9) at 0, 10, 20, 30, 40 and 50mV steps compared with LQTS1 cardiomyocytes without treatment ( n =10). ( g ) Representative epi-fluorescent and phase-contrast images of LQTS1 cardiomyocytes with and without Dxm treatment from proximity ligation assay (PLA, SIGMAR1-hERG, red, DAPI, blue). Scale bar, 10μm. ( h ) SIGMAR1-hERG PLA quantification in Dxm-treated ( n =20) and non-treated ( n =20) LQTS1 cardiomyocytes. ( i ) Representative epi-fluorescent and phase-contrast images of LQTS1 cardiomyocytes with and without Dxm treatment from PLA (SIGMAR1-K V 7.1, red, DAPI, blue). Scale bar, 10μm. ( j ) SIGMAR1-K V 7.1 PLA quantification in Dxm-treated ( n =13) and non-treated ( n =23) LQTS1 cardiomyocytes. All data are mean ± s.d. The treatment of Fluvo or Dxm for the experiments was 5μM, 2hrs. One-way ANOVA with Tukey’s multiple comparisons was used for b, and unpaired two-tailed Student’s t -test was used for h, j between the groups and for d, f at each voltage step. * P <0.05, ** P <0.01, *** P <0.001, n.s., not significant. The cardiomyocytes were from at least two independent differentiations.

**Techniques Used: **Derivative Assay, Proximity Ligation Assay, Two Tailed Test

**Figure Legend Snippet:**( a ) Representative traces of action potentials in long QT syndrome type 2 (LQTS2) iPSC-derived cardiomyocytes without treatment or treated with fluvoxamine (+Fluvo) or dextromethorphan (+Dxm). ( b ) Action potential duration (APD90) analysis in LQTS2 cardiomyocytes without treatment ( n =11) or treated with fluvoxamine ( n =10) or dextromethorphan ( n =11). Other action potential parameters are shown in , , . ( c ) Representative traces of I Kr currents (E-4031-sensitive) in LQTS2 cardiomyocytes treated with Dxm or without treatment. I Kr currents (steady-state) were significantly increased in Dxm-treated LQTS2 cardiomyocytes ( n =9) at −10 and 0mV steps compared with LQTS2 cardiomyocytes without treatment ( n =10). ( e ) Representative traces of I Ks currents (Chromanol-293B-sensitive) in LQTS2 cardiomyocytes treated with Dxm or without treatment. I Ks currents (steady-state) were significantly increased in Dxm-treated LQTS2 cardiomyocytes at 0, 10, 20, 30 and 50mV steps compared with LQTS2 cardiomyocytes without treatment ( n =10/group). ( g ) Representative epi-fluorescent and phase-contrast images of LQTS2 cardiomyocytes with and without Dxm treatment from proximity ligation assay (PLA, SIGMAR1-hERG, red, DAPI, blue). Scale bar, 10μm. ( h ) SIGMAR1-hERG PLA quantification in Dxm-treated ( n =22) and non-treated ( n =29) LQTS2 cardiomyocytes. ( i ) Representative epi-fluorescent and phase-contrast images of LQTS2 cardiomyocytes with and without Dxm treatment from PLA (SIGMAR1-K V 7.1, red, DAPI, blue). Scale bar, 10μm. ( j ) SIGMAR1-K V 7.1 PLA quantification in Dxm-treated ( n =107) and non-treated ( n =87) LQTS2 cardiomyocytes. All data are mean ± s.d. The treatment of Fluvo or Dxm for the experiments was 5μM, 2hrs. One-way ANOVA with Tukey’s multiple comparisons was used for b. Unpaired two-tailed Student’s t -test was used for h, j between the groups and for d, f at each voltage step. * P <0.05, ** P <0.01, *** P <0.001, n.s., not significant. The cardiomyocytes were from at least two independent differentiations.

**Techniques Used: **Derivative Assay, Proximity Ligation Assay, Two Tailed Test

**Figure Legend Snippet:**( a-f ) Analysis of APD50 (50% from peak, a, b ), peak amplitude ( c, d ) and resting potential ( e, f ) in LQTS1 cardiomyocytes without treatment ( n =11) or treated with fluvoxamine (Fluvo, n =10) or dextromethorphan (Dxm, n =10) and in LQTS2 cardiomyocytes without treatment ( n =11) or treated with Fluvo ( n =10) or Dxm ( n =11). ( g,h ) I Kr current (tail) was significantly increased by Dxm in LQTS1 cardiomyocytes at 40mV step ( g, LQTS1, n =10; +Dxm, n =9) and in LQTS2 cardiomyocytes at 10, 30 and 40mV steps ( h, LQTS2, n =10; +Dxm, n =9). ( i,j ) Representative traces of Ba 2+ currents in LQTS1 ( i ) or LQTS2 cardiomyocytes( j ) without treatment or treated with Dxm. ( k,l ) Voltage-dependent calcium channel inactivation was not significantly changed by Dxm in LQTS1( k ) or LQTS2 cardiomyocytes ( l ) compared to non-treated cells ( n =10/group). ( m,n ) Current-voltage relationship of Ba 2+ recordings in Dxm-treated ( n =10) and non-treated LQTS1 cardiomyocytes ( n =7)( m ) and in Dxm-treated ( n =7) and non-treated LQTS2 cardiomyocytes ( n =8)( n ). ( o,p ) Representative immunoblots of human SIGMAR1, ATF4, CDK5, CDK5R1/p35 and GAPDH protein using lysates from control (Ctrl) cardiomyocytes, LQTS1 cardiomyocytes ( o ) and LQTS2 cardiomyocytes ( p ). ^, p35 antibody used for was discontinued (Santa Cruz, sc-820). Therefore, another antibody (Cell Signaling Technology, C64B10) was used for this blotting series while its signal-to-noise ratio was not as high as sc-820. ( q-s ) Quantification of SIGMAR1 25kDa protein band ( q, r ) and # 35kDa protein band ( s , possibly a dimer of a full-length SIGMAR1 with a SIGMAR1 splice variant ) in LQTS1 or LQTS2 cardiomyocytes compared to Ctrl ( n =4/group). ( t,u ) Quantification of CDK5 (left) and CDK5R1/p35 protein expressions (right) in LQTS1 or LQTS2 cardiomyocytes compared to control cardiomyocytes ( n =4/group). ( v,w ) Quantification of ATF4 38kDa protein band (left, non-modified), 50kDa (center, phosphorylated) and 70kDa protein expression (right, ubiquitinated) in LQTS1 or LQTS2 cardiomyocytes compared to control cardiomyocytes ( n =4/group). All data are mean ± s.d. The treatment of Dxm or Fluvo for the experiments was 5μM, 2hrs. One-way ANOVA with Tukey’s multiple comparisons was used for a,b,c,d,e,f. Unpaired two-tailed Student’s t -test was used for k,l,q,r,s,t,u,v,w between the groups and for g,h,m,n at each voltage step. * P <0.05, ** P <0.01, n.s., not significant. The cardiomyocytes were from at least two independent differentiations.

**Techniques Used: **Western Blot, Variant Assay, Modification, Expressing, Two Tailed Test

# sigmar1 (Alomone Labs)

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## Images

### 1) Product Images from "Sigma non-opioid receptor 1 is a potential therapeutic target for long QT syndrome"

**Article Title: **Sigma non-opioid receptor 1 is a potential therapeutic target for long QT syndrome

**Journal: **Nature cardiovascular research

**doi: **10.1038/s44161-021-00016-2

**Figure Legend Snippet:**( a ) Schematic representation of altered molecules (red) and therapeutic strategy using SIGMAR1 agonists (blue) in Timothy syndrome (TS) cardiomyocytes. The bottom table shows the characteristics of SIGMAR1 agonists. OCD, obsessive compulsive disorder. PBA, Pseudo Bulbar Affect. SSRI, selective serotonin reuptake inhibitor. NMDAR, N -Methyl-D-aspartate receptor. ( b ) Docking model of SIGMAR1 and its agonist PRE-084. Representative traces of 0.2Hz-paced action potentials in TS cardiomyocytes without treatment or treated with SIGMAR1 agonists, PRE-084 (+PRE), fluvoxamine (+Fluvo) or dextromethorphan (+Dxm). ( d ) Action potential duration (APD90, 90% reduction from peak) analysis in TS cardiomyocytes without treatment ( n =15) or treated with PRE ( n =13), Fluvo ( n =10) and Dxm ( n =11). Other action potential parameters are shown in – . ( e ) Representative traces of 0.5Hz-paced action potentials in isogenic control (Ctrl) iPSC-derived cardiomyocytes without treatment or treated with Dxm or Fluvo. APD90 analysis in the control cells without treatment ( n =10) or treated with Dxm ( n =11) or Fluvo ( n =10). Other action potential parameters of isogenic Ctrl are shown in – . All data are mean ± s.d. The treatment of PRE, Fluvo and Dxm for the experiments was 5μM, 2hrs. One-way ANOVA with Tukey’s multiple comparisons was used for d, f. ** P <0.01, n.s., not significant. The cardiomyocytes were from at least two independent differentiations.

**Techniques Used: **Derivative Assay

**Figure Legend Snippet:**( a ) Docking models of SIGMAR1-Fluvo, -Dxm and -PRE. ( b ) PRE-084 (5μM, 2hr) reduced CDK5 kinase activity in Timothy syndrome (TS) cardiomyocytes. PHA-793887 (PHA, 5μM), a CDK5 inhibitor, was used as positive control for the assay ( n =8/group). ( c-e ) The effects of PRE (5μM, 2hr) on CDK5R1/p35 protein ( c, d, n =12 for baseline and n =9 for 2 hr after treatment) and CDK5 protein ( c, d, n =5/group) and mRNA ( e, n =4/group). ( f ) Representative epi-fluorescent and phase-contrast images of TS cardiomyocytes without treatment, with PRE-084 (+PRE, 5μM, 2hr) or PRE-084 and NE-100 treatment (+PRE & NE-100, both 5μM, 2hr) from proximity ligation assay (PLA, SIGMAR1-CDK5, red, DAPI, blue). Scale bar, 10μm. ( g ) SIGMAR1-CDK5 PLA quantification in PRE-treated ( n =16), PRE&NE-100-treated ( n =38) and non-treated TS cardiomyocytes ( n =14). ( h ) Representative traces of relative motion analysis of TS cardiomyocyte contractions before (black) and after 2-hr PRE-084 treatment (blue). The representative traces were obtained from and . ( i-j ) Relative changes of beating rate ( i ) and irregularity ( j ) ( n =11) of TS cardiomyocytes after PRE-084 treatment. ( k ) Representative traces of Ba 2+ currents in TS cardiomyocytes without treatment or treated with PRE-084 (+PRE), or fluvoxamine (+Fluvo) (each, 5μM, 2hr) and in isogenic control cardiomyocytes (Ctrl). +Dxm representative trace is shown in . ( l ) Voltage-dependent calcium channel inactivation was significantly enhanced by PRE ( n =10), Fluvo ( n =10) and Dxm ( n =16) treatment in TS cardiomyocytes compared to non-treated cells ( n =25). n.s., no significant differences between +PRE, +Fluvo, +Dxm and isogenic Ctrl groups ( n =13). ( m ) Representative traces of Ba 2+ currents in TS cardiomyocytes before treatment and acutely treated with PRE (5μM, ~5–10 mins). ( n ) Voltage-dependent calcium channel inactivation was not significantly changed by acute PRE treatment in TS cardiomyocytes ( n =10). All data are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons was used for b, g and One-way ANOVA with Sidak’s multiple comparisons was used for l. Paired two-tailed Student’s t -test was used for i, j, n, and unpaired two-tailed Student’s t -test was used for d, e. * P <0.05, ** P <0.01, *** P <0.001, n.s., not significant. Cell samples from at least two independent differentiations were used.

**Techniques Used: **Activity Assay, Positive Control, Proximity Ligation Assay, Two Tailed Test

**Figure Legend Snippet:**( a-c ) Action potential parameters, APD50 ( a ), resting potential ( b ) and peak amplitude ( c ) in Timothy syndrome (TS) cardiomyocytes without treatment ( n =15) or treated for 2 hrs with 5μM SIGMAR1 agonists, PRE-084 (+PRE, n =13), fluvoxamine (+Fluvo, n =10) or dextromethorphan (+Dxm, n =11). APD90 is shown in . ( d-f ) Action potential parameters, APD50 ( d ), resting potential ( e ) and peak amplitude ( f ) in isogenic control (Ctrl) cardiomyocytes without treatment ( n =10) and treated for 2 hrs with 5μM SIGMAR1 agonists, Fluvo ( n =10) or Dxm ( n =11). ( g ) Quantification of human SIGMAR1 transcripts (normalized to GAPDH) in Timothy syndrome ( n =9 from two independent lines) and control cardiomyocytes ( n =12 from four independent lines). ( h ) Representative immunoblots of human SIGMAR1 and GAPDH protein using lysates from TS and control iPSC-derived cardiomyocytes. ( i-j ) Quantification of SIGMAR1 25kDa protein band ( i ) and 35kDa protein expression ( j , normalized to GAPDH) in TS iPSC-derived cardiomyocytes compared to the isogenic Ctrl ( n =6/group). The molecular weight of SIGMAR1 is ~25 kDa while the 35kDa band ( # ) has been reported previously and might be a dimer of a full-length SIGMAR1 with a SIGMAR1 splice variant . ( k ) Representative immunoblots of human ATF4 using the same lysates from TS and isogenic Ctrl iPSC-derived cardiomyocytes shown in . ( l-n ) Quantification of ATF4 38kDa protein band ( l , non-modified), 50kDa ( m , phosphorylated) and 70kDa protein expression ( n , ubiquitinated, normalized to GAPDH) in TS iPSC-derived cardiomyocytes compared to the isogenic Ctrl ( n =6/group). ( o-r ) ATF4 overexpression ( o ) significantly increased SIGMAR1 transcription ( p ) and protein expression ( q,r ) in normal human cardiomyocytes transfected with ATF4 plasmid (+ ATF4, o&p, n =7, r, n =5). The cardiomyocytes were harvested 24 hr after the lipofection. The empty vector was used as a negative control (Mock, o&p, n =5, r, n =4). ( q ) Representative immunoblots of human SIGMAR1 and GAPDH using the lysate from the transfected cardiomyocytes. All data are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons was used for a-f and unpaired two-tailed Student’s t -test was used for g, i, j, l, m, n, o, p, r. * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001, n.s., not significant. Cell samples from at least two independent differentiations were used.

**Techniques Used: **Western Blot, Derivative Assay, Expressing, Molecular Weight, Variant Assay, Modification, Over Expression, Transfection, Plasmid Preparation, Negative Control, Two Tailed Test

**Figure Legend Snippet:**( a-b ) Representative traces of time-course calcium imaging in spontaneously contracting isogenic control (Ctrl, black) and Timothy syndrome (TS, red) cardiomyocytes. ( c-d ) Calcium transient frequency ( c ) and duration ( d ) analysis in TS cardiomyocytes during the 2-hr imaging ( n =32). ( e ) Current-voltage relationship of Ba 2+ recordings in Dxm-treated ( n =12) and non-treated TS cardiomyocytes ( n =13). ( f ) Representative traces of Ba 2+ currents in isogenic control cardiomyocytes without treatment (Ctrl) or treated with dextromethorphan (+Dxm). ( g ) Voltage-dependent calcium channel inactivation was not altered by Dxm ( n =10) in the isogenic control cardiomyocytes compared to non-treated cells ( n =13). ( h ) Representative epi-fluorescent and phase-contrast images of TS cardiomyocytes without and with Dxm (+Dxm) from proximity ligation assay (PLA, SIGMAR1-Ca V 1.2, red, DAPI, blue). Scale bar, 10μm. ( i ) SIGMAR1-Ca V 1.2 PLA quantification in Dxm-treated ( n =33) and non-treated TS cardiomyocytes (TS, n =32). ( j ) I Kr current steady-state amplitudes were significantly reduced in TS cardiomyocytes ( n =10) compared to isogenic control ( n =10) at −10, 0 and 10 mV steps from −40mV hold. ( k ) I Kr tail currents were significantly reduced in TS cardiomyocytes ( n =10) compared to isogenic control ( n =10) at −10, 0, 10 and 20 mV steps. ( l ) Representative traces of I Kr currents (E-4031-sensitive) in isogenic Ctrl cardiomyocytes (black) and TS cardiomyocytes treated with Dxm (purple), Dxm & NE-100 (green) or without treatment (red, TS). The I Kr traces are shown in . ( m ) There was no significant difference in I Kr current (tail) between Dxm-treated ( n =9) and non-treated TS cardiomyocytes ( n =10) while Dxm & NE-100 ( n =10) significantly reduced tail currents compared to Dxm at 10, 20, 30, 40 and 50 mV steps and also to non-treated TS at 20mV step from −40mV hold. All data are mean ± s.d. The treatment of Dxm or NE-100 for all experiments was 5μM, 2hrs. One-way ANOVA with Tukey’s multiple comparisons was used for c, d between the time points to before and used for m at each voltage step. Unpaired two-tailed Student’s t -test were used for g, i between the groups and used for e, j, k at each voltage step. * P <0.05, n.s., no significant. The samples were from at least two independent differentiations.

**Techniques Used: **Imaging, Proximity Ligation Assay, Two Tailed Test

**Figure Legend Snippet:**( a ) Representative traces of time-course calcium imaging in spontaneously contracting Timothy syndrome (TS) cardiomyocytes treated with Dxm (5μM, until 120min). ( b-c ) Calcium transient frequency ( b ) and duration( c ) analysis in TS cardiomyocytes before and after Dxm treatment ( n =19). ( d ) Representative traces of Ca 2+ currents in TS cardiomyocytes with and without Dxm. ( e ) Late calcium current analysis in TS cardiomyocytes with and without Dxm (TS, n =11; +Dxm, n =12). ( f ) Representative traces of Ba 2+ currents in TS cardiomyocytes without treatment, with Dxm (5μM, 2hrs, +Dxm) or Dxm with a SIGMAR1 antagonist, NE-100 (1μM, +Dxm&NE-100). ( g ) Voltage-dependent inactivation in TS cardiomyocytes without treatment ( n =25), with Dxm ( n =16) or Dxm+NE-100 ( n =11). ( h ) Representative traces of I Kr currents (E-4031-sensitive) in TS cardiomyocytes with Dxm (5μM), Dxm&NE-100 (each, 5μM) or without treatment. ( i ) I Kr current amplitude analysis in TS cardiomyocytes with Dxm ( n =9), Dxm&NE-100 ( n =10) or without treatment ( n =10) (* P <0.05, Dxm vs TS and Dxm vs Dxm & NE-100, at −10, 0, 10, 20 and 30mV). ( j ) Representative confocal fluorescent and bright-field images of TS cardiomyocytes without and with Dxm treatment (+Dxm) from proximity ligation assay (PLA, SIGMAR1-hERG, red, DAPI, blue). Scale bar, 10μm. ( k ) SIGMAR1-hERG PLA quantification in Dxm-treated ( n =36) and non-treated ( n =35) TS cardiomyocytes. ( l ) Representative traces of I Ks currents (Chromanol-293B-sensitive) in TS cardiomyocytes treated with Dxm (5μM), Dxm&NE-100 (each, 5μM) or without treatment. ( m ) I Ks current amplitude analysis in TS cardiomyocytes with Dxm ( n =10), Dxm&NE-100 ( n =9) or without treatment ( n =10) (* P <0.05, Dxm vs TS and Dxm vs Dxm & NE-100, at −10, 0, 10, 20, 30 and 40mV). ( n ) Representative epi-fluorescent and phase-contrast images of TS cardiomyocytes without and with Dxm treatment (+Dxm) from PLA (SIGMAR1-K V 7.1, red, DAPI, blue). Scale bar, 10μm. ( o ) SIGMAR1-K V 7.1 PLA quantification in Dxm-treated ( n =40) and non-treated ( n =20) TS cardiomyocytes. All data are mean ± s.d. One-way ANOVA with Tukey’s multiple comparisons was used for b, c, g between the groups and for i, m at each voltage step. Unpaired two-tailed Student’s t -test was used for e, k, o. * P <0.05, ** P <0.01, *** P <0.001, n.s., not significant. The samples were from at least two independent differentiations.

**Techniques Used: **Imaging, Proximity Ligation Assay, Two Tailed Test

**Figure Legend Snippet:**( a ) Representative traces of I Kr current (E-4031-sensitive) in isogenic control (Ctrl) cardiomyocytes treated with Dxm (5μM, 2hr) or without treatment. ( b-c ) I Kr current amplitude ( b ) and tail I Kr current ( c ) were not significantly changed by Dxm in the isogenic Ctrl cardiomyocytes ( n =10/group). ( d ) Schematic representation of confocal imaging for proximity ligation assay (PLA, SIGMAR1-hERG, red) in human iPSC-derived cardiomyocytes. ( e ) Representative confocal fluorescent images of TS cardiomyocytes used for PLA (SIGMAR1-hERG, red) and DAPI (blue) staining. Dxm treatment (+Dxm, 5μM, 2hr) was conducted to examine the effect of Dxm on PLA signals in TS cardiomyocytes. #3 images are used in . Scale bar, 10μm. ( f ) Representative epi-fluorescent and phase-contrast images of isogenic Ctrl cardiomyocytes used for PLA (red, SIGMAR1-hERG) and DAPI (blue) staining. Dxm treatment (+Dxm, 5μM, 2hr) was conducted to examine the effect of Dxm on PLA signals in the cardiomyocytes. Scale bar, 10μm. ( g ) Quantification of PLA signal number of SIGMAR1-hERG in Dxm-treated ( n =21) and non-treated ( n =21) isogenic Ctrl cardiomyocytes. ( h ) Representative traces of I Ks current (Chromanol 293B-sensitive) in isogenic control (Ctrl) cardiomyocytes treated with Dxm or without treatment. ( i ) I Ks current amplitude analysis in the isogenic Ctrl cardiomyocytes with Dxm (5μM, 2hr, n =9) or without treatment ( n =10). ( j ) Representative epi-fluorescent and phase-contrast images of isogenic Ctrl cardiomyocytes used for PLA (red, SIGMAR1-K V 7.1) and DAPI (blue) staining. Dxm treatment (+Dxm, 5μM, 2hr) was conducted to examine the effect of Dxm on PLA signals in the cardiomyocytes. Scale bar, 10μm. ( k ) Quantification of PLA signal number of SIGMAR1-K V 7.1 in Dxm-treated ( n =40) and non-treated ( n =44) isogenic Ctrl cardiomyocytes. All data are mean ± s.d. Unpaired two-tailed Student t -test was used for g,k between the groups, and for b,c,i at each voltage step. * P <0.05. n.s., not significant. The cell samples were from at least two independent differentiations.

**Techniques Used: **Imaging, Proximity Ligation Assay, Derivative Assay, Staining, Two Tailed Test

**Figure Legend Snippet:**( a ) Representative traces of action potentials in long QT syndrome type 1 (LQTS1) iPSC-derived cardiomyocytes without treatment or treated with fluvoxamine (+Fluvo) or dextromethorphan (+Dxm). ( b ) Action potential duration (APD90) analysis in LQTS1 cardiomyocytes without treatment ( n =11) or treated with fluvoxamine ( n =10) or dextromethorphan ( n =10). Other action potential parameters are shown in , , . Representative traces of I Ks currents (Chromanol-293B-sensitive) in LQTS1 cardiomyocytes treated with Dxm or without treatment. ( d ) I Ks currents (steady-state) were significantly increased by Dxm in LQTS1 cardiomyocytes at all voltage steps ( n =10/group). Representative traces of I Kr currents (E-4031-sensitive) in LQTS1 cardiomyocytes treated with Dxm or without treatment. ( f ) I Kr currents (steady-state) were significantly increased in Dxm-treated LQTS1 cardiomyocytes ( n =9) at 0, 10, 20, 30, 40 and 50mV steps compared with LQTS1 cardiomyocytes without treatment ( n =10). ( g ) Representative epi-fluorescent and phase-contrast images of LQTS1 cardiomyocytes with and without Dxm treatment from proximity ligation assay (PLA, SIGMAR1-hERG, red, DAPI, blue). Scale bar, 10μm. ( h ) SIGMAR1-hERG PLA quantification in Dxm-treated ( n =20) and non-treated ( n =20) LQTS1 cardiomyocytes. ( i ) Representative epi-fluorescent and phase-contrast images of LQTS1 cardiomyocytes with and without Dxm treatment from PLA (SIGMAR1-K V 7.1, red, DAPI, blue). Scale bar, 10μm. ( j ) SIGMAR1-K V 7.1 PLA quantification in Dxm-treated ( n =13) and non-treated ( n =23) LQTS1 cardiomyocytes. All data are mean ± s.d. The treatment of Fluvo or Dxm for the experiments was 5μM, 2hrs. One-way ANOVA with Tukey’s multiple comparisons was used for b, and unpaired two-tailed Student’s t -test was used for h, j between the groups and for d, f at each voltage step. * P <0.05, ** P <0.01, *** P <0.001, n.s., not significant. The cardiomyocytes were from at least two independent differentiations.

**Techniques Used: **Derivative Assay, Proximity Ligation Assay, Two Tailed Test

**Figure Legend Snippet:**( a ) Representative traces of action potentials in long QT syndrome type 2 (LQTS2) iPSC-derived cardiomyocytes without treatment or treated with fluvoxamine (+Fluvo) or dextromethorphan (+Dxm). ( b ) Action potential duration (APD90) analysis in LQTS2 cardiomyocytes without treatment ( n =11) or treated with fluvoxamine ( n =10) or dextromethorphan ( n =11). Other action potential parameters are shown in , , . ( c ) Representative traces of I Kr currents (E-4031-sensitive) in LQTS2 cardiomyocytes treated with Dxm or without treatment. I Kr currents (steady-state) were significantly increased in Dxm-treated LQTS2 cardiomyocytes ( n =9) at −10 and 0mV steps compared with LQTS2 cardiomyocytes without treatment ( n =10). ( e ) Representative traces of I Ks currents (Chromanol-293B-sensitive) in LQTS2 cardiomyocytes treated with Dxm or without treatment. I Ks currents (steady-state) were significantly increased in Dxm-treated LQTS2 cardiomyocytes at 0, 10, 20, 30 and 50mV steps compared with LQTS2 cardiomyocytes without treatment ( n =10/group). ( g ) Representative epi-fluorescent and phase-contrast images of LQTS2 cardiomyocytes with and without Dxm treatment from proximity ligation assay (PLA, SIGMAR1-hERG, red, DAPI, blue). Scale bar, 10μm. ( h ) SIGMAR1-hERG PLA quantification in Dxm-treated ( n =22) and non-treated ( n =29) LQTS2 cardiomyocytes. ( i ) Representative epi-fluorescent and phase-contrast images of LQTS2 cardiomyocytes with and without Dxm treatment from PLA (SIGMAR1-K V 7.1, red, DAPI, blue). Scale bar, 10μm. ( j ) SIGMAR1-K V 7.1 PLA quantification in Dxm-treated ( n =107) and non-treated ( n =87) LQTS2 cardiomyocytes. All data are mean ± s.d. The treatment of Fluvo or Dxm for the experiments was 5μM, 2hrs. One-way ANOVA with Tukey’s multiple comparisons was used for b. Unpaired two-tailed Student’s t -test was used for h, j between the groups and for d, f at each voltage step. * P <0.05, ** P <0.01, *** P <0.001, n.s., not significant. The cardiomyocytes were from at least two independent differentiations.

**Techniques Used: **Derivative Assay, Proximity Ligation Assay, Two Tailed Test

**Figure Legend Snippet:**( a-f ) Analysis of APD50 (50% from peak, a, b ), peak amplitude ( c, d ) and resting potential ( e, f ) in LQTS1 cardiomyocytes without treatment ( n =11) or treated with fluvoxamine (Fluvo, n =10) or dextromethorphan (Dxm, n =10) and in LQTS2 cardiomyocytes without treatment ( n =11) or treated with Fluvo ( n =10) or Dxm ( n =11). ( g,h ) I Kr current (tail) was significantly increased by Dxm in LQTS1 cardiomyocytes at 40mV step ( g, LQTS1, n =10; +Dxm, n =9) and in LQTS2 cardiomyocytes at 10, 30 and 40mV steps ( h, LQTS2, n =10; +Dxm, n =9). ( i,j ) Representative traces of Ba 2+ currents in LQTS1 ( i ) or LQTS2 cardiomyocytes( j ) without treatment or treated with Dxm. ( k,l ) Voltage-dependent calcium channel inactivation was not significantly changed by Dxm in LQTS1( k ) or LQTS2 cardiomyocytes ( l ) compared to non-treated cells ( n =10/group). ( m,n ) Current-voltage relationship of Ba 2+ recordings in Dxm-treated ( n =10) and non-treated LQTS1 cardiomyocytes ( n =7)( m ) and in Dxm-treated ( n =7) and non-treated LQTS2 cardiomyocytes ( n =8)( n ). ( o,p ) Representative immunoblots of human SIGMAR1, ATF4, CDK5, CDK5R1/p35 and GAPDH protein using lysates from control (Ctrl) cardiomyocytes, LQTS1 cardiomyocytes ( o ) and LQTS2 cardiomyocytes ( p ). ^, p35 antibody used for was discontinued (Santa Cruz, sc-820). Therefore, another antibody (Cell Signaling Technology, C64B10) was used for this blotting series while its signal-to-noise ratio was not as high as sc-820. ( q-s ) Quantification of SIGMAR1 25kDa protein band ( q, r ) and # 35kDa protein band ( s , possibly a dimer of a full-length SIGMAR1 with a SIGMAR1 splice variant ) in LQTS1 or LQTS2 cardiomyocytes compared to Ctrl ( n =4/group). ( t,u ) Quantification of CDK5 (left) and CDK5R1/p35 protein expressions (right) in LQTS1 or LQTS2 cardiomyocytes compared to control cardiomyocytes ( n =4/group). ( v,w ) Quantification of ATF4 38kDa protein band (left, non-modified), 50kDa (center, phosphorylated) and 70kDa protein expression (right, ubiquitinated) in LQTS1 or LQTS2 cardiomyocytes compared to control cardiomyocytes ( n =4/group). All data are mean ± s.d. The treatment of Dxm or Fluvo for the experiments was 5μM, 2hrs. One-way ANOVA with Tukey’s multiple comparisons was used for a,b,c,d,e,f. Unpaired two-tailed Student’s t -test was used for k,l,q,r,s,t,u,v,w between the groups and for g,h,m,n at each voltage step. * P <0.05, ** P <0.01, n.s., not significant. The cardiomyocytes were from at least two independent differentiations.

**Techniques Used: **Western Blot, Variant Assay, Modification, Expressing, Two Tailed Test

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