Journal: Biochimica et Biophysica Acta

Article Title: Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons

doi: 10.1016/j.bbamcr.2013.02.012

Figure Lengend Snippet: Identification of RyR2 as a binding partner of RTN1A. (A) Schematic representation of full length RTN1A and the GST-RTN1 523 construct ( right ). RHD: reticulon homology domain; TM1 and TM2: transmembrane domain 1 and 2. GST pull-downs were performed with GST-RTN1 523 using detergent-solubilized mouse brain proteins. GST or empty glutathione beads served as negative controls, whereas GST-NiR was tested to control for GST-RTN1 523 binding specificity. Arrow denotes protein band that was consistently pulled down with GST-RTN1 523 and from which RyR2 was identified by mass spectrometry. Note that this band is absent in the different control samples. Asterisks indicate the GST fusion proteins and their relative amounts used in the GST pull-down. The silver stained gel shown is representative of three independent experiments. (B) Upper panel , Western Blot of samples from (A) probed with RyR2 antibody. Note the double-band that is identified as RyR2. The higher molecular mass band corresponds to intact RyR2, while the lower band presumably represents a proteolytic degradation fragment of RyR2. The intensity of the RyR2 bands increase with increasing amounts of GST-RTN1 523 . Lower panel , shows Ponceau S staining of the pull-downs to assess the relative amounts of each GST fusion protein. Input lane shows one-twentieth of the amount used for pull-down. WB, Western blot. The Western blot shown is representative of three independent experiments.

Article Snippet: RyR2 was identified with a mouse antibody recognizing all RyR isoforms (1:1000; Pierce), RyR1 with rabbit anti-RyR1 (1:1000; Alomone Labs, Israel), IP3R1 with rabbit anti-IP3R1 (1:4000; Alomone Labs, Israel) and RTN4A with either rabbit or mouse anti-RTN4A .

Techniques: Binding Assay, Construct, Control, Mass Spectrometry, Staining, Western Blot

Journal: Biochimica et Biophysica Acta

Article Title: Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons

doi: 10.1016/j.bbamcr.2013.02.012

Figure Lengend Snippet: RTN1A associates preferentially with RyR2 channel in vivo . (A) Left panels , detergent-solubilized protein from HEK293 cells transiently transfected with untagged RyR2 plus RTN1A-myc was immunoprecipitated (IP) with rabbit polyclonal anti-RTN1A antibodies or control rabbit IgG. Immunoprecipitated proteins were detected on immunoblots with monoclonal anti-RyR2 or anti-RTN1A antibodies. Note that native HEK293 cells do not express endogenous levels of RTN1A or RyR2. Right panels , detergent-solubilized protein from HEK293 cells transiently transfected with untagged RyR2 plus RTN4A-myc was immunoprecipitated with rabbit polyclonal anti-RTN4 antibodies or control rabbit IgG. Immunoprecipitated proteins were detected on immunoblots with monoclonal anti-RyR2 or anti-RTN4A antibodies. Input lane shows one-eighth of the amount used for immunoprecipitation. WB, Western blot. (B) Detergent-solubilized protein from rat cerebellum was used for co-immunoprecipitations with rabbit anti-RTN1A, rabbit anti-RTN1A, or control rabbit IgG. Immunoprecipitated proteins were resolved by SDS-PAGE blotted on PVDF membranes and probed with monoclonal antibodies as indicated at the right. Input lane shows one-tenth of the amount used for immunoprecipitation. WB, Western blot. (C) Detergent-solubilized protein from rat cerebellum was used for co-immunoprecipitations with mouse anti-RyR2, mouse anti-RTN1A, mouse anti-RTN1A, or control mouse IgG. Immunoprecipitated proteins were resolved by SDS-PAGE, blotted on PVDF membranes and probed with antibodies as indicated at the right. Input lane shows one-tenth of the amount used for immunoprecipitation. WB, Western blot. (D) Detergent-solubilized protein from rat cerebellum was immunoprecipitated with mouse anti-RTN1A, mouse anti-RyR2, or control mouse IgG. Immunoprecipitated proteins were resolved by SDS-PAGE, blotted on PVDF membranes and probed with antibodies as indicated at the right. Note that RyR1 co-immunoprecipitates with mouse anti-RyR2, but not with mouse anti-RTN1A antibodies. Input lane shows one-fifth of the amount used for immunoprecipitation. WB, Western blot.

Article Snippet: RyR2 was identified with a mouse antibody recognizing all RyR isoforms (1:1000; Pierce), RyR1 with rabbit anti-RyR1 (1:1000; Alomone Labs, Israel), IP3R1 with rabbit anti-IP3R1 (1:4000; Alomone Labs, Israel) and RTN4A with either rabbit or mouse anti-RTN4A .

Techniques: In Vivo, Transfection, Immunoprecipitation, Control, Western Blot, SDS Page, Bioprocessing

Journal: Biochimica et Biophysica Acta

Article Title: Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons

doi: 10.1016/j.bbamcr.2013.02.012

Figure Lengend Snippet: Immunocytochemical distribution of mCherry-RTN1 523 and RyRs in HEK293 cells. (A) HEK293 cells were transiently transfected with mCherry-RTN1 523 in the absence (a) or presence of untagged RyR2 (d–f) or RyR1 (g–i). Single-transfections of untagged RyR2 (b) and RyR1 (c) were carried out as a comparison. Cells were immunostained with anti-RyR2 (b,d–f) or anti-RyR1 (c,g-i) antibodies and visualized by confocal microscopy. Note that single-transfected mCherry-RTN1 523 is uniformly distributed in the cytosol (a), but altered to a more reticular staining pattern when co-expressed with RyR2 (d–f). mCherry-RTN1 523 remained uniformly distributed in cells cotransfected with RyR1 (g–i). Cells shown represent at least 20 representative cells per condition. (B) Immunofluorescence confocal microscopy analysis of RyR ER localization in HEK293 cells. HEK293 cells were single-transfected with untagged RyR1 cDNA or RyR2 cDNA, immunostained with the indicated antibodies and imaged using immunofluorescence microscopy to demonstrate RyR and calreticulin (ER-specific marker) colocalization. Cells were immunostained for RyR isoforms (green) and Calreticulin (red), respectively. Space bar: 10 μm. (C) Extent of colocalization between mCherry-RTN1 523 and RyR isoforms was quantified using Pearson correlation coefficient and determined through correlation analysis with Leica SP5 software from 10 different cells per group. (*** p = 0.003 by Student's t -test).

Article Snippet: RyR2 was identified with a mouse antibody recognizing all RyR isoforms (1:1000; Pierce), RyR1 with rabbit anti-RyR1 (1:1000; Alomone Labs, Israel), IP3R1 with rabbit anti-IP3R1 (1:4000; Alomone Labs, Israel) and RTN4A with either rabbit or mouse anti-RTN4A .

Techniques: Transfection, Comparison, Confocal Microscopy, Staining, Immunofluorescence, Microscopy, Marker, Software

Journal: Biochimica et Biophysica Acta

Article Title: Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons

doi: 10.1016/j.bbamcr.2013.02.012

Figure Lengend Snippet: Identification of the RyR2 binding domain. (A) Schematic representation of rat RTN1A protein and RTN1A fragments used to construct GST fusion proteins for the pull-down experiments. HHD: high homology domain; LNT: long N-terminal fragment. (B) GST-RTN1 fragments were used as baits in pull-down experiments using detergent-solubilized mouse brain proteins. GST, GST-NiR or empty glutathione beads served as negative controls. Binding of RyR2 was subsequently detected by immunoblot ( upper panel ). Ponceau S staining of the pull-downs shows the relative amounts of each GST fusion protein ( lower panel ). Input lane shows one-tenth of the amount used for immunoprecipitation. WB, Western blot. Results are representative of three independent experiments.

Article Snippet: RyR2 was identified with a mouse antibody recognizing all RyR isoforms (1:1000; Pierce), RyR1 with rabbit anti-RyR1 (1:1000; Alomone Labs, Israel), IP3R1 with rabbit anti-IP3R1 (1:4000; Alomone Labs, Israel) and RTN4A with either rabbit or mouse anti-RTN4A .

Techniques: Binding Assay, Construct, Western Blot, Staining, Immunoprecipitation

Journal: Biochimica et Biophysica Acta

Article Title: Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons

doi: 10.1016/j.bbamcr.2013.02.012

Figure Lengend Snippet: Immunohistochemical distribution pattern of RTN1A and RyR2 in rat hippocampus and cerebellum. Representative staining patterns for RTN1A (A,C,D) and RyR2 (B,E) on sections of rat hippocampus (A,B) and cerebellar cortex (C,D,E). In the hippocampus, immunoreactivity for both proteins is found in granule cells (G), mossy fiber axons, and in stratum lucidum (SL). In the cerebellum, RTN1A-immunoreactivity was confined to Purkinje cell bodies (P), their dendrites in ML (C; arrow) and axons (D; arrowheads). (E) Confocal immunofluorescent image showing RyR2 staining in Purkinje cells. Unlike RTN1A, RyR2 was also found in granule cell layer (Gl). Scale bars: A and B, 500 μm; H, hilus; G, Granule cell layer; So, stratum oriens; Sr, stratum radiatum; Slm, stratum lacunosum molecular; Iml, inner molecular layer; M + Oml, Middle & outer molecular layer; CA1-3, Cornu ammonis; SL, stratum lucidum; S, Subiculum; Pr, Presubiculum; Pa, Parasubiculum.

Article Snippet: RyR2 was identified with a mouse antibody recognizing all RyR isoforms (1:1000; Pierce), RyR1 with rabbit anti-RyR1 (1:1000; Alomone Labs, Israel), IP3R1 with rabbit anti-IP3R1 (1:4000; Alomone Labs, Israel) and RTN4A with either rabbit or mouse anti-RTN4A .

Techniques: Immunohistochemical staining, Staining

Journal: Biochimica et Biophysica Acta

Article Title: Direct association of the reticulon protein RTN1A with the ryanodine receptor 2 in neurons

doi: 10.1016/j.bbamcr.2013.02.012

Figure Lengend Snippet: GST-RTN1 523 inhibits specific [ 3 H]ryanodine binding to rat brain synaptosomes. (A) Equilibrium [ 3 H]ryanodine binding to rat forebrain synaptosomal membrane preparations was carried out in binding buffer containing 10 nM [ 3 H]ryanodine at the indicated free calcium concentrations in control conditions ( closed circles ) and in the presence of 0.1 μM GST ( closed triangles ) or 0.1 μM GST-RTN1 523 ( open squares ). [Ca 2 + ] was maintained, in the range 0.01 μM–1 mM, by a combination of EGTA and CaCl 2 . Free Ca 2 + concentrations were calculated as described in material and methods. Data points shown are the mean ± S.E.M., from three separate experiments performed in triplicates. (B) [ 3 H]ryanodine binding in the presence of 0.1 μM GST or GST-RTN1 523 is presented as percent of control. No specific [ 3 H]ryanodine binding was observed at 0.01 μM Ca 2 + in the presence of GST or GST-RTN1 523 . Difference in [ 3 H]ryanodine binding was plotted as percent decrease in specific binding. Data points shown are the mean ± S.E.M., from three separate experiments (* p = 0.011 by Student's t -test). (C) RyR2 evoked Ca 2 + oscillations in HEK293. Upper and lower left panels represent Fura-2 ratio time-courses of single cells expressing RyR2 together with mcherry, mcherry-RTN1A or EGFP-RTN4A. Cells were continuously perfused with buffer containing 0 mM Ca 2 + (nominal free), 1 mM Ca 2 + and 0 mM Ca 2 + + 10 mM caffeine as indicated by the bars at the top. Lower right panel shows a quantitative analysis performed by integration of the respective single peak areas referred to area under curve for estimation of the total amount of the cytosolic [Ca 2 + ] arising through RyR2 dependent Ca 2 + oscillations. The fraction of cells that showed both oscillations as well as a clear caffeine peak in comparison to those that lacked oscillations before a single caffeine peak are given in percentages in the graph. A two-sample t-test was carried out to test for significance as indicated by the p values at the bottom of the panel.

Article Snippet: RyR2 was identified with a mouse antibody recognizing all RyR isoforms (1:1000; Pierce), RyR1 with rabbit anti-RyR1 (1:1000; Alomone Labs, Israel), IP3R1 with rabbit anti-IP3R1 (1:4000; Alomone Labs, Israel) and RTN4A with either rabbit or mouse anti-RTN4A .

Techniques: Binding Assay, Membrane, Control, Expressing, Comparison

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Evidence of functional ryanodine receptors in rat mesenteric collecting lymphatic vessels

doi: 10.1152/ajpheart.00564.2018

Figure Lengend Snippet: Blockade of ryanodine receptors (RyRs) with ryanodine leads to a loss of phasic contractions and limited substance P (SP)-induced contraction. Representative trace of diameter over time of an isolated rat collecting mesenteric lymphatic vessel treated with 10−5 M ryanodine (A). The continuation of the trace shows a later time period in the same experiment with ryanodine, when 10−8 M SP was added. The boxes beneath the trace represent the time periods during which the quantitative data used for comparisons in B–D were collected. The mean diameter (B), mean tone (C), and maximum tone (D) for the time periods before addition of SP, 0–30 s after SP addition, and 31–150 s after addition of SP were compared using a repeated measures ANOVA model followed by Dunnett’s comparison with control (baseline period). P values for the comparisons are shown. N = 6 lymphatics. Each lymphatic studied was obtained from a unique rat. NS, not significant.

Article Snippet: The primer sequences used, obtained from Applied Biosystems/Thermo Fisher Scientific, were Rn01545085_m1 (Ryr1), Rn01470303_m1 (Ryr2), Rn01486097_m1 (Ryr3), and Rn01775763_g1 (GAPDH).

Techniques: Isolation

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Evidence of functional ryanodine receptors in rat mesenteric collecting lymphatic vessels

doi: 10.1152/ajpheart.00564.2018

Figure Lengend Snippet: The ryanodine receptor (RyR)1/RyR3 inhibitor dantrolene does not inhibit rat mesenteric collecting lymphatic contractions or the increased CF and decreased EDD/Max elicited by substance P (SP). Representative trace of diameter over time of an isolated rat mesenteric collecting lymphatic vessel before and after the addition of 10−5 M dantrolene, which remained in the bath (A). Continuation of the same trace, showing the addition of 10−8 M SP in the presence of dantrolene (B). The boxes beneath the traces in A and B indicate 2-min time periods during which the quantitative data used for comparisons in C–H were collected. Comparisons between baseline (BL), after addition of dantrolene, and after addition of SP in the presence of dantrolene were performed for CF (C), EDD/MaxD (D), ESD/MaxD (E), AMP/MaxD (F), EF (G), and FPF (H). P values are shown for comparisons that were found to be significantly different (P < 0.05) using Tukey’s multiple comparison test after an initial one-way repeated measures ANOVA. N = 6 isolated lymphatics studied from 6 different rats. CF, contraction frequency; EDD, end-diastolic diameter; EF, ejection fraction; ESD, end-systolic diameter; FPF, fractional pump flow; MaxD, maximal passive diameter; NS, not significant.

Article Snippet: The primer sequences used, obtained from Applied Biosystems/Thermo Fisher Scientific, were Rn01545085_m1 (Ryr1), Rn01470303_m1 (Ryr2), Rn01486097_m1 (Ryr3), and Rn01775763_g1 (GAPDH).

Techniques: Isolation

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Evidence of functional ryanodine receptors in rat mesenteric collecting lymphatic vessels

doi: 10.1152/ajpheart.00564.2018

Figure Lengend Snippet: Quantitative PCR (qPCR) detection of ryanodine receptors (RyRs) in rat mesenteric collecting lymphatic vessels. A: means show relative expression to GAPDH determined by qPCR and using the 2-ΔCt method in isolated rat mesenteric collecting lymphatics (N = 7 rats) and rat brain, skeletal muscle, and cardiac muscle (all N = 2 rats). B: gel of the single amplicon products of expected size from the qPCR reactions. Rat mesenteric collecting lymphatics were harvested (30 lymphatic vessels per rat), and total RNA (50 ng) was reverse transcribed into cDNA and qPCR performed with specific primer/probe sets for Ryr1 (Rn01545085_m1), Ryr2 (Rn01470303_m1), Ryr3 (Rn01486097_m1), and GAPDH (Rn01775763_g1). Reactions with no template were also run as controls (NTC). The gel shown is representative of three separate experiments using mesenteric lymphatic samples obtained from three different rats.

Article Snippet: The primer sequences used, obtained from Applied Biosystems/Thermo Fisher Scientific, were Rn01545085_m1 (Ryr1), Rn01470303_m1 (Ryr2), Rn01486097_m1 (Ryr3), and Rn01775763_g1 (GAPDH).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Isolation, Amplification

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Evidence of functional ryanodine receptors in rat mesenteric collecting lymphatic vessels

doi: 10.1152/ajpheart.00564.2018

Figure Lengend Snippet: Determination of ryanodine receptor (RyR)2 and RyR3 localization in isolated rat mesenteric collecting lymphatic vessels with immunofluorescence labeling and laser confocal microscopy. A. maximum intensity z-projection (37 confocal slices) of a lymphatic vessel labeled with anti-RyR2 AlexaFluor488-conjugated secondary antibodies and DAPI. The RyR2 labeling appeared mostly in hoop-like patterns in the vessel wall, with some labeling in the adventitial layer (1 example denoted by the small white arrow). B: maximum intensity z-projection (85 confocal slices) of a lymphatic immunolabeled for RyR3 and nuclei. A similar hoop-like pattern was observed with RyR3 immunolabeling, with some adventitial layer labeling (small white arrow). No signal was detected in labeling controls with no primary antibody (data not shown). C and E: labeling of RyR2, smooth muscle actin (SMA), nuclei, and a merge of the three channels in a single confocal slice featuring a cross-section of the collecting lymphatic wall. The orientation is with the adventitial layer on the left and the endothelial layer on the right. RyR2 labeling was predominantly found in the smooth muscle layer, as evidenced by partial colocalization with SMA and RyR2 labeling surrounding the nuclei of smooth muscle cells. D and F: labeling of RyR3, SMA, nuclei, and a merge of the three channels in a single confocal slice featuring a cross-section of the lymphatic wall, oriented with the adventitial layer on the left and endothelium on the right. RyR3 was predominantly found in smooth muscle cells, as evidenced by the partial colocalization with SMA. The small arrows in E and F point toward nuclei of endothelial cells (ECs). A and B: representative of N = 8 labeling experiments each. C–F: representative of N = 4 experiments each. The scale bars in C–F represent 5 µm.

Article Snippet: The primer sequences used, obtained from Applied Biosystems/Thermo Fisher Scientific, were Rn01545085_m1 (Ryr1), Rn01470303_m1 (Ryr2), Rn01486097_m1 (Ryr3), and Rn01775763_g1 (GAPDH).

Techniques: Isolation, Immunofluorescence, Labeling, Confocal Microscopy, Immunolabeling

Journal: Aging (Albany NY)

Article Title: An aging and p53 related marker: HOXA5 promoter methylation negatively correlates with mRNA and protein expression in old age

doi: 10.18632/aging.202621

Figure Lengend Snippet: DNAm of the target CpG sites of DUSP3, HOXA5 and RYR1. Mean DNA methylation values of the differentially methylated CpG sites are demonstrated in young and old subjects. The associated genes were assigned to the target CpG site, respectively. Detection of mean DNA methylation was performed by microarray on bisulfite-converted DNA prepared from monocytes. Error bars denote SEM (n = 4, average age: 23.8 yrs and 81.4 yrs, respectively). P values are FDR adjusted.

Article Snippet: qRT-PCR for mRNA detection was performed using 10 μl 2x Taqman Universal PCR Master Mix II and 1 μl Taqman gene expression assay ( HOXA5 - Cat No. Hs00430330_m1, DUSP3 - Cat No. Hs01115776_m1, RYR1 - Cat No. Hs01062613_m1, p53 - Cat No. Hs01034249_m1 and PTEN - Cat No. Hs02621230_s1, Thermo Fisher Scientific), 8 μl RNase-free water and 1 μl cDNA template (50 ng/μl).

Techniques: DNA Methylation Assay, Methylation, Microarray

Journal: Aging (Albany NY)

Article Title: An aging and p53 related marker: HOXA5 promoter methylation negatively correlates with mRNA and protein expression in old age

doi: 10.18632/aging.202621

Figure Lengend Snippet: Deep analysis of CpG sites. ( A ) Validated methylation values of the microarray-detected CpG sites are demonstrated in young and old subjects. Detection of mean DNA methylation was performed by local deep sequencing on bisulfite-converted DNA prepared from monocytes. The associated genes are assigned to the target CpG site, respectively. Error bars denote SEM (n = 20, average age: 23.4 and 79.6 yrs, respectively). ( B – D ) shows the correlation between the methylation of the target CpG site of DUSP3 , HOXA5 and RYR1 and the participant's age. Methylation values of the 8 individuals that were originally applied in the MethylationEPIC BeadChip Microarray are accentuated by red dots. ( B ) Depicts the correlation between the methylation of the target CpG site of DUSP3 and the participants age showing a very low correlation coefficient (r = -0.0255). ( C ) Delineates a linear correlation between HOXA5 methylation in the target CpG site of the HOXA5 promoter region and the participants age showing increasing methylation levels with increasing age as reflected in the respective correlation coefficient (r = 0.7437). ( D ) Depicts the correlation between the methylation of the target CpG site of RYR1 and and the participants age showing a very low correlation coefficient (r = 0.1174).

Article Snippet: qRT-PCR for mRNA detection was performed using 10 μl 2x Taqman Universal PCR Master Mix II and 1 μl Taqman gene expression assay ( HOXA5 - Cat No. Hs00430330_m1, DUSP3 - Cat No. Hs01115776_m1, RYR1 - Cat No. Hs01062613_m1, p53 - Cat No. Hs01034249_m1 and PTEN - Cat No. Hs02621230_s1, Thermo Fisher Scientific), 8 μl RNase-free water and 1 μl cDNA template (50 ng/μl).

Techniques: Methylation, Microarray, DNA Methylation Assay, Sequencing

Journal: Aging (Albany NY)

Article Title: An aging and p53 related marker: HOXA5 promoter methylation negatively correlates with mRNA and protein expression in old age

doi: 10.18632/aging.202621

Figure Lengend Snippet: Age- dependent HOXA5 mRNA decline . mRNA expression levels of DUSP3 , HOXA5 and RYR1 are demonstrated in young and old subjects. While there was no significant difference in mRNA expression demonstrated for DUSP3 and RYR1 , HOXA5 mRNA levels are significantly down-regulated in monocytes of old blood donors. Detection of mRNA expression was performed by qRT-PCR on total cellular RNA prepared from monocytes. Error bars denote SEM (n = 30, average age: 23.0 yrs and 81.1 yrs, respectively).

Article Snippet: qRT-PCR for mRNA detection was performed using 10 μl 2x Taqman Universal PCR Master Mix II and 1 μl Taqman gene expression assay ( HOXA5 - Cat No. Hs00430330_m1, DUSP3 - Cat No. Hs01115776_m1, RYR1 - Cat No. Hs01062613_m1, p53 - Cat No. Hs01034249_m1 and PTEN - Cat No. Hs02621230_s1, Thermo Fisher Scientific), 8 μl RNase-free water and 1 μl cDNA template (50 ng/μl).

Techniques: Expressing, Quantitative RT-PCR

Journal: Human mutation

Article Title: RyR1 deficiency in congenital myopathies disrupts excitation-contraction coupling.

doi: 10.1002/humu.22326

Figure Lengend Snippet: Figure 1. Protein expression of RyR1 and DHPR in muscle biopsy of patients with different RYR1 mutations. Double staining of RyR1 and DHPR, and NADH staining in muscle biopsies of patients with congenital myopathy and RYR1 mutations. NADH staining was performed in non-serial sections from RyR1/DHPR double staining. Scale bar = 25 µm.

Article Snippet: RYR1 siRNA (Santa Cruz Biotechnology) was transfected using lipofectamine 2000 in OptiMEM medium following the manufacturer’s recommendations (Invitrogen).

Techniques: Expressing, Double Staining, Staining

Journal: Human mutation

Article Title: RyR1 deficiency in congenital myopathies disrupts excitation-contraction coupling.

doi: 10.1002/humu.22326

Figure Lengend Snippet: Figure 2. Quantitative reverse transcriptional real-time PCR of RYR1 and CACNA1S in skeletal muscle biopsies from patients carrying different RYR1 mutations. The relative content of RYR1 and CACNA1S mRNA was assessed by real-time PCR using Ct method and using DES as muscle specific reference gene. The relative expression of RYR1 and CACNA1S to DES in one of the control individuals was set as 1.0, and was used to calibrate the values of all other samples. Each symbol represents an individual and the bar indicates mean relative expression. ANOVA was used for the statistical analysis of the values in different groups, followed by the Bonferroni post statistics test. A: Relative expression of RYR1 transcript in muscle biopsies. B: Relative expression of CACNA1S transcript in muscle biopsies. ∗P < 0.05, ∗∗P < 0.01.

Article Snippet: RYR1 siRNA (Santa Cruz Biotechnology) was transfected using lipofectamine 2000 in OptiMEM medium following the manufacturer’s recommendations (Invitrogen).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Control

Journal: Human mutation

Article Title: RyR1 deficiency in congenital myopathies disrupts excitation-contraction coupling.

doi: 10.1002/humu.22326

Figure Lengend Snippet: Figure 3. RYR1, CACNA1S, and DES mRNA expression in myotubes treated with RYR1 siRNA. The relative quantification of RYR1 (A), CACNA1S (B), and DES (C) mRNA was performed by quantitative real-time PCR. The value was obtained from three independent transfection experiments of myotubes treated with RYR1 siRNA at concentrations of 10, 30, and 50 nM. The data are presented as mean ± SEM, N = 3. ANOVA was used for the statistical analysis of the values in the RYR1 siRNA-treated myotubes to negative control siRNA, followed by Bonferroni post statistics test. The relative expression of target genes in control siRNA-treated myotubes was set as 1.0, and was used to normalize the values in the RYR1 siRNA-treated myotubes. ∗P < 0.05, ∗∗P < 0.01.

Article Snippet: RYR1 siRNA (Santa Cruz Biotechnology) was transfected using lipofectamine 2000 in OptiMEM medium following the manufacturer’s recommendations (Invitrogen).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Negative Control, Control

Journal: Human mutation

Article Title: RyR1 deficiency in congenital myopathies disrupts excitation-contraction coupling.

doi: 10.1002/humu.22326

Figure Lengend Snippet: Figure 4. Downregulation of RyR1 by RYR1 siRNA transfection does not affect myotube differentiation: Cells were transfected with 50 nM RYR1 siRNA, differentiated for 5 days and then stained with β-tubulin and DAPI, the diameter of myotubes with >3 nuclei were measured and averagedconsidered. A: Mean (±SE) myotube diameter (µm) of 30 transfected and 30 mock transfected myotubes. (B: Relative mRNA expression of differentiation-related markers; Dystroglycan 1 (DAG), Myosin heavy chain 1 (MYH1), Troponin T1 (TNNT1), and Desmin (DES) normalized to GAPDH expression as internal control (N = 6). C: Immunofluorescentce of transfected (RYR1siRNA) or mock transfected (control) myotubes stained with anti- β tubulin (green) and DAPI and observed with a Nikon A1R confocal microscope with a 40× NeoFluar objective (1.4 NA). Bar indicates 30 µm.

Article Snippet: RYR1 siRNA (Santa Cruz Biotechnology) was transfected using lipofectamine 2000 in OptiMEM medium following the manufacturer’s recommendations (Invitrogen).

Techniques: Transfection, Staining, Expressing, Control, Microscopy

Journal: Human mutation

Article Title: RyR1 deficiency in congenital myopathies disrupts excitation-contraction coupling.

doi: 10.1002/humu.22326

Figure Lengend Snippet: Figure 5. RyR1, DHPR, and other SR protein in RYR1 siRNA-treated myotubes and muscle biopsies from patients with RyR1 deficiency due to recessive RYR1 mutations. A: Representative western blots of RyR1, DHPR, IP3R-III, α-actinin 2, SERCA 2, desmin, and β-tubulin in human myotubes treated by RYR1 siRNA. B: Semi-quantification of proteins in RYR1 siRNA-treated myotubes. The expression of proteins was normalized to tubulin. Protein content in RYR1 siRNA-treated group was compared with the control group. C: Representative western blots of RyR1, DHPR, IP3R-III, α-actinin 2, SERCA 2, and desmin in muscle biopsies from two patients (patient 2 and 5 in Supp. Table S1) with recessive RYR1 mutations. D: Semi-quantification of proteins in two patients, with RyR1 deficiency due to recessive RYR1 mutations, and three controls. The expression of proteins was normalized by desmin.

Article Snippet: RYR1 siRNA (Santa Cruz Biotechnology) was transfected using lipofectamine 2000 in OptiMEM medium following the manufacturer’s recommendations (Invitrogen).

Techniques: Western Blot, Expressing, Control

Journal: Human mutation

Article Title: RyR1 deficiency in congenital myopathies disrupts excitation-contraction coupling.

doi: 10.1002/humu.22326

Figure Lengend Snippet: Figure 6. The expression of ITPR1, ITPR2, and ITPR3 mRNA in RYR1 siRNA-treated myotubes and skeletal muscle biopsies from patients with different RYR1 mutations. A: The relative expression of ITPRs mRNA in cultured myotubes treated with siRNA was measured by quantitative reverse transcript real-time PCR. Data are presented as mean ± SEM. N = 4 samples per group. ∗P < 0.05. B: The relative expression of ITPRs mRNA measured by quantitative real-time PCR in skeletal muscle biopsies from controls (N = 7), patients with dominant RYR1 mutations (N = 5) and patients with complex recessive RYR1 mutations with RyR1 protein deficiency (N = 6). GAPDH was used as general internal control gene. C: The relative expression of ITPRs mRNA measured by quantitative real-time PCR in skeletal muscle biopsies by using DES as muscle-specific internal control gene.

Article Snippet: RYR1 siRNA (Santa Cruz Biotechnology) was transfected using lipofectamine 2000 in OptiMEM medium following the manufacturer’s recommendations (Invitrogen).

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Control

Journal: Human mutation

Article Title: RyR1 deficiency in congenital myopathies disrupts excitation-contraction coupling.

doi: 10.1002/humu.22326

Figure Lengend Snippet: Figure 7. Analysis of calcium regulation in a human muscle cell line after transfection with RYR1 siRNA. Cells were transfected with 50 nM RYR1 siRNA or mock transfected as described in the Materials and Methods section and were either untreated or treated with 1 µM Xestospongin C during the 40 min of fura-2 loading. Myotubes were stimulated with the indicated agonist in Krebs-Ringer containing no added Ca2+ plus 100 µM La3+ and the total amount of calcium released was calculated using Origin software by calculating the total transient, i.e. the area under the curve. Resting [Ca2+] is presented as fluorescence ratio (340/380 nm) (au) before cell stimulation in Krebs-Ringer containing 2 mM Ca2+; the total amount of rapidly releasable Ca2+ present in the SR/ER stores was obtained by calculating the area under the curve after exposing cells to 1 µM ionomycin in the Krebs-Ringer containing no added Ca2+ plus 0.5 mM EGTA. For details see Materials and Methods section. All results are expressed as mean value (±SEM) of the indicated number of cells. White bars mock transfected cells, grey bars cells transfected with RYR1 siRNA. Statistical analysis was performed using Student’s t-test.

Article Snippet: RYR1 siRNA (Santa Cruz Biotechnology) was transfected using lipofectamine 2000 in OptiMEM medium following the manufacturer’s recommendations (Invitrogen).

Techniques: Transfection, Software, Cell Stimulation