Journal: Frontiers in Molecular Neuroscience

Article Title: Expression of genes encoding the calcium signalosome in cellular and transgenic models of Huntington's disease

doi: 10.3389/fnmol.2013.00042

Figure Lengend Snippet: List of genes and their annotations included in the RT-qPCR array .

Article Snippet: Ryr3 , ryanodine receptor 3 , Mm01328421_m1.

Techniques: Binding Assay

Journal: Nature Communications

Article Title: Cellular and molecular landscape of mammalian sinoatrial node revealed by single-cell RNA sequencing

doi: 10.1038/s41467-020-20448-x

Figure Lengend Snippet: a qPCR shows the expression of Ryr3 (Cluster 1), Apold1 (Cluster 2), and Col1a1 (Cluster 3) in sinoatrial node (SAN), atrial, and ventricular tissue, respectively ( n = 3 biologically independent animals per group). Dunnett’s multiple comparisons test, data are represented as mean ± s.e.m., adjusted p value was labeled on the top. RA right atrium, LA left atrium, RV right ventricle, LV, left ventricle. b – d Immunostaining shows the location of RYR3 ( b ), APOLD1 ( c ), and COL1A1 ( d ) in mouse SAN tissue, respectively. Representative images are shown from n = 3 biologically independent samples. Scale bar = 50 μm. Zoom images (the box regions in the merged images) show the higher magnification. Scale bar = 10 μm.

Article Snippet: Primary antibodies included HCN4 (Sigma, SAB5200035, 1:50), Connexin 43 (CST, 3512, 1:50), VSNL1 (Gene Tex, GTX115039, 1:50), Collagen I (Abcam, ab21286, 1:50), DLGAP1 (Affbiotech, AF0308, 1:50), UNC80 (BIOSS, BS-12121R, 1:50), APOLD1 (Novus Biologicals, NBP2-58460, 1:50), RYR3 (Novus Biologicals, NBP2-76962, 1:50), Connexin 40 (Invitrogen, 37-8900, 1:50), cTNT (Abcam, ab8295, 1:50).

Techniques: Expressing, Labeling, Immunostaining

Journal: Cancer science

Article Title: miR-367 promotes proliferation and stem-like traits in medulloblastoma cells.

doi: 10.1111/cas.12733

Figure Lengend Snippet: Fig. 5. Downregulation of miR-367 cancer-related targets in medul- loblastoma cells. (a) Relative amount of cells with positive expression of RYR3, assessed by flow cytometry 48 h post-transfection with either miR-367 mimic or non-specific control. D283-Med cells were negative for RYR3 expression. (b) RAB23 protein levels in medulloblastoma cells, assessed by western blotting 48 h post-transfection with either miR-367 mimic or non-specific control. Respective blot quantification is presented as a bar graph. Significance level: *P < 0.05, **P < 0.01, ****P < 0.0001.

Article Snippet: After 48 h of cellular transfection, cells were permeabilized with the FIX & PERM Cell Fixation and Cell Permeabilization Kit (Life Technologies) and incubated with anti-RYR3 antibody (NPB1-70399; Novus Biologicals, Littleton, CO).

Techniques: Expressing, Cytometry, Transfection, Control, Western Blot

Journal: American Journal of Physiology - Cell Physiology

Article Title: Purinergic agonists increase [Ca 2+ ] i in rat conjunctival goblet cells through ryanodine receptor type 3

doi: 10.1152/ajpcell.00291.2024

Figure Lengend Snippet: Effect of blocking L-type calcium channels with nifedipine and ryanodine receptors with dantrolene on ATP and BzATP-induced peak [Ca 2+ ] i . Cultured rat conjunctival goblet cells were preincubated with nifedipine (Nif) (10 −5 M) for 15 min and/or dantrolene (Da) (10 −5 M) for 30 min before stimulation with ATP (10 −5 M) ( A ) or BzATP (10 −4 M) ( B ). A and B : the average [Ca 2+ ] i over time. Arrows represent the addition of agonists. C : the peak increase of [Ca 2+ ] i above baseline. ATP: bars to the left of the central vertical dotted line in C , BzATP: bars to the right of the central vertical dotted line in C . Data are presented as means ± SE. n = 3. BzATP, benzoylbenzoyl-ATP; [Ca 2+ ] i , intracellular calcium concentration. *Statistically significant difference from agonist alone.

Article Snippet: Cells were incubated with monoclonal mouse anti-RyR1/2 antibody (Thermo Fisher Scientific, Cat. No. MA3-916, RRID:AB_2183054) or rabbit anti-RyR3 antibody (Alomone Labs, Cat. No. ARR-003, RRID:AB_2040186) at a 1:50 or 1:100 dilution overnight at 4°C.

Techniques: Blocking Assay, Cell Culture, Concentration Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Purinergic agonists increase [Ca 2+ ] i in rat conjunctival goblet cells through ryanodine receptor type 3

doi: 10.1152/ajpcell.00291.2024

Figure Lengend Snippet: RT-qPCR of the ryanodine receptor 3 (RyR3) subtype in rat and human conjunctival goblet cells (CGCs). Total RNA was isolated from rat and human CGCs and reverse-transcribed. cDNA amplification confirmed the presence of RyR3 mRNA in rat ( A ) but not in human ( B ) CGCs. Relative amount of RyR3 transcript compared with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was measured by quantitative PCR and is shown on y -axis. n = 3. RT-qPCR, quantitative reverse transcription polymerase chain reaction. *Statistically significant difference from 0.

Article Snippet: Cells were incubated with monoclonal mouse anti-RyR1/2 antibody (Thermo Fisher Scientific, Cat. No. MA3-916, RRID:AB_2183054) or rabbit anti-RyR3 antibody (Alomone Labs, Cat. No. ARR-003, RRID:AB_2040186) at a 1:50 or 1:100 dilution overnight at 4°C.

Techniques: Quantitative RT-PCR, Isolation, Reverse Transcription, Amplification, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

Journal: American Journal of Physiology - Cell Physiology

Article Title: Purinergic agonists increase [Ca 2+ ] i in rat conjunctival goblet cells through ryanodine receptor type 3

doi: 10.1152/ajpcell.00291.2024

Figure Lengend Snippet: Proposed regulation of intracellular calcium levels by ryanodine receptor 3 (RyR3) in cultured rat conjunctival goblet cells. ATP and BzATP may increase [Ca 2+ ] i : 1 ) directly by activating RyR3, 2 ) by activating purinergic 1 (P1) receptors after the enzymatic conversion to metabolites, 3 ) by the activation of purinergic 2 (P2) receptors directly, and 4 ) by activating L-type calcium channels through membrane depolarization. These mechanisms can then either directly increase [Ca 2+ ] i through the influx of extracellular Ca 2+ and/or through the activation of RyR3 on an intracellular compartment such as the ER to release Ca 2+ . BzATP, benzoylbenzoyl-ATP; [Ca 2+ ] i , intracellular calcium concentration; CGCs, conjunctival goblet cells; CICR, calcium-induced calcium release.

Article Snippet: Cells were incubated with monoclonal mouse anti-RyR1/2 antibody (Thermo Fisher Scientific, Cat. No. MA3-916, RRID:AB_2183054) or rabbit anti-RyR3 antibody (Alomone Labs, Cat. No. ARR-003, RRID:AB_2040186) at a 1:50 or 1:100 dilution overnight at 4°C.

Techniques: Cell Culture, Activation Assay, Membrane, Concentration Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Purinergic agonists increase [Ca 2+ ] i in rat conjunctival goblet cells through ryanodine receptor type 3

doi: 10.1152/ajpcell.00291.2024

Figure Lengend Snippet: Identification of ryanodine receptors (RyRs) in rat and human conjunctival goblet cells (CGCs). Immunofluorescence microscopy was used to determine the presence of RyR3 ( A ) or RyR1/2 ( B ) from rat ( middle ) and human ( right ) CGCs. Negative controls ( left ) are representative for rat and human cells. Red: RyR3, green: RyR1/2, blue: DAPI. Images are representative for at least three individuals. Scale bars are located in the upper right corner of all micrographs. Scale bar = 25–75 μm.

Article Snippet: Cells were incubated with monoclonal mouse anti-RyR1/2 antibody (Thermo Fisher Scientific, Cat. No. MA3-916, RRID:AB_2183054) or rabbit anti-RyR3 antibody (Alomone Labs, Cat. No. ARR-003, RRID:AB_2040186) at a 1:50 or 1:100 dilution overnight at 4°C.

Techniques: Immunofluorescence, Microscopy, Negative Control

Journal: Frontiers in Cellular Neuroscience

Article Title: Mediation of Autophagic Cell Death by Type 3 Ryanodine Receptor (RyR3) in Adult Hippocampal Neural Stem Cells

doi: 10.3389/fncel.2016.00116

Figure Lengend Snippet: A list of primers used for quantitative real-time PCR analysis .

Article Snippet: Guide RNAs for gene inactivation of RyR3 were designed and purchased from ToolGen (Seoul, Republic of Korea).

Techniques: Real-time Polymerase Chain Reaction

Journal: Frontiers in Cellular Neuroscience

Article Title: Mediation of Autophagic Cell Death by Type 3 Ryanodine Receptor (RyR3) in Adult Hippocampal Neural Stem Cells

doi: 10.3389/fncel.2016.00116

Figure Lengend Snippet: Genetic suppression of RyR3 diminishes autophagic flux in I(−) HCN cells. (A–C) Effect of CRIPR/Cas9-induced RYR3 knockout on ACD in I(−) HCN cells. (A) An experimental scheme for inhibition of autophagic flux by BafA1 treatment. (B) Characterization of autophagic flux by immunoblotting of LC3 and SQSTM1/p62 in RYR3KO I(−) HCN cells treated with BafA1. Cells were treated with BafA1 for 2 h at given concentration before harvest. (C) Rate of cell death in RYR3KO HCN cells under I(+) and I(−) conditions for 24 h, 48 h, and 72 h; each experiment performed in triplicate per experiment ( n = 4). The bars represent the mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001; n.s., non-significant.

Article Snippet: Guide RNAs for gene inactivation of RyR3 were designed and purchased from ToolGen (Seoul, Republic of Korea).

Techniques: Knock-Out, Inhibition, Western Blot, Concentration Assay

Journal: Frontiers in Cellular Neuroscience

Article Title: Mediation of Autophagic Cell Death by Type 3 Ryanodine Receptor (RyR3) in Adult Hippocampal Neural Stem Cells

doi: 10.3389/fncel.2016.00116

Figure Lengend Snippet: Type 3 Ryanodine receptor (RyR) is the major isoform in HCN cells and its expression is upregulated following insulin withdrawal. (A) Real-time quantitative PCR analysis of basal expression levels of RyR and IP 3 R genes denoted as RYR and ITPR, respectively, after normalization to β-actin. (B,C) A change in the mRNA levels of RYR (B) and ITPR (C) 24 h following insulin withdrawal. Each set of experiment was conducted in triplicate per experiment ( n = 4). The bars represent the mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Guide RNAs for gene inactivation of RyR3 were designed and purchased from ToolGen (Seoul, Republic of Korea).

Techniques: Expressing, Real-time Polymerase Chain Reaction

Journal: Frontiers in Cellular Neuroscience

Article Title: Mediation of Autophagic Cell Death by Type 3 Ryanodine Receptor (RyR3) in Adult Hippocampal Neural Stem Cells

doi: 10.3389/fncel.2016.00116

Figure Lengend Snippet: ER Ca 2+ efflux is significantly inhibited in I(−) RYR3KO HCN cells. (A,B) Caffeine was used to provoke ER-to-cytosol transfer of Ca 2+ in control I(−) and RYR3KO I(−) HCN cells. (A) Immunofluorescence images of pCMV R-CEPIA1er in HCN cells experiencing autophagy depicted by puncta formation of EGFP-LC3 24 h following insulin withdrawal. Cells subjected to analysis are indicated by arrowheads. Scale bar, 10 μm. Fluorescence intensity of CEPIA1er (B) selectively in EGFP-LC3-positive HCN cells was quantified ( n = 36 for Cas9 Control I(−), 41 for Cas9 Control I(−)/Caffeine, 32 for RYR3KO I(−), 43 for RYR3KO/Caffeine). The bars represent the mean ± SEM; * p < 0.05, *** p < 0.001; n.s., non-significant. (C,D) Caffeine failed to facilitate ACD of I(−) HCN cells in the absence of RyR3. (C) Western blot analysis of cleaved caspase-3 and LC3 in I(−) RYR3KO HCN cells treated with caffeine. (D) Cell death analysis in RYR3KO HCN cells treated with caffeine for 24 h. Each experiment set was performed in triplicate per experiment ( n = 6). The bars represent the mean ± SEM; ** p < 0.01, *** p < 0.001; n.s., non-significant.

Article Snippet: Guide RNAs for gene inactivation of RyR3 were designed and purchased from ToolGen (Seoul, Republic of Korea).

Techniques: Control, Immunofluorescence, Fluorescence, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: Mediation of Autophagic Cell Death by Type 3 Ryanodine Receptor (RyR3) in Adult Hippocampal Neural Stem Cells

doi: 10.3389/fncel.2016.00116

Figure Lengend Snippet: Autophagic flux in RYR3KO HCN cells is unaltered upon insulin withdrawal or caffeine treatment. (A,B) Caffeine failed to induce autophagic flux of I(−) HCN cells in the absence of RyR3. (A) Immunofluorescence analysis of HCN cells transfected with mRFP-GFP-LC3 tandem construct. Images were analyzed 24 h following insulin withdrawal with or without caffeine treatment. Cells subjected to analysis are indicated by arrowheads. Scale bar, 10 μm. (B) Quantification of mRFP- and GFP-LC3 puncta in RYR3KO HCN cells treated with caffeine compared to controls ( n = 22 for Cas9 Control I(+), 30 for Cas9 Control I(−), 27 for Cas9 Control I(−)/Caffeine, 29 for RYR3KO I(+), 34 for RYR3KO I(−), 25 for RYR3KO/Caffeine). The bars represent the mean ± SEM; * p < 0.05, ** p < 0.01, *** p < 0.001; n.s., non-significant. The black and red asterisks indicate the statistical significance in the comparison of total and mRFP puncta numbers, respectively.

Article Snippet: Guide RNAs for gene inactivation of RyR3 were designed and purchased from ToolGen (Seoul, Republic of Korea).

Techniques: Immunofluorescence, Transfection, Construct, Control, Comparison

Journal: Frontiers in Cellular Neuroscience

Article Title: Mediation of Autophagic Cell Death by Type 3 Ryanodine Receptor (RyR3) in Adult Hippocampal Neural Stem Cells

doi: 10.3389/fncel.2016.00116

Figure Lengend Snippet: Schematic diagram illustrating the role of ER-to-cytosol Ca 2+ in regulation of survival and death of NSCs in insulin withdrawal model. Presence of insulin constitutively ensures proper distribution of intracellular Ca 2+ which maintains homeostasis of HCN cells. Inactivation of the pro-survival insulin signaling pathway by insulin withdrawal leads to increased expression and activation of RyR3 which subsequently discharge Ca 2+ from the ER, the major intracellular Ca 2+ store. Increase in cytosolic Ca 2+ subsequently triggers a signaling cascade that induces ACD in HCN cells.

Article Snippet: Guide RNAs for gene inactivation of RyR3 were designed and purchased from ToolGen (Seoul, Republic of Korea).

Techniques: Expressing, Activation Assay