Journal: Journal of Neuroscience

Article Title: Interactive HIV-1 Tat and Morphine-Induced Synaptodendritic Injury Is Triggered through Focal Disruptions in Na+ Influx, Mitochondrial Instability, and Ca2+ Overload

doi: 10.1523/jneurosci.5351-13.2014

Figure Lengend Snippet: Figure6. Effectsofmorphineand/orTaton[Ca 2]iand[Na ]iinneuronaldendritesfollowing0–30minexposure.A,AcuteTatmorphine-inducedincreasesin[Ca 2]iareattenuatedby ryanodineorpyruvate,whilenimodipine(L-typeCa 2channelblocker)anddantrolenedonotshowanyeffectsonTatmorphine-inducedchangesin[Ca 2]i.B,Average[Ca 2]iduringthefinal 10min(from20to30min)indicateryanodinesignificantlyblockscombinedTatandmorphine-inducedincreasesin[Ca 2]i,whereasnoeffectsarenotedfornimodipine,dantrolene,orpyruvate. C, Ca 2-free medium or low [Na ]o significantly decreases Tat plus morphine increases in [Ca 2]i. However, Ca 2-free or low Na medium significantly decreases [Ca 2]i following Tat treatment alone, but Tat increases are exacerbated after 20 min. D, Tat morphine-induced increases in [Ca 2]i during the interval from 20 to 30 min are significantly reduced by Ca 2-free medium.E,Tatmorphinesimilarlyincreases[Na ]i,whichiscompletelyblockedbylow[Na ]o.AlthoughCa 2-freemediumattenuatestheTatmorphine-associatedelevationsin[Na ]i, [Na ]i levels remained markedly above baseline levels perhaps suggesting some Ca 2 efflux from internal stores. Exogenous pyruvate also significantly attenuate Tat morphine-induced increasesin[Na ]i,whereasryanodinehasnoeffect.F,Average[Na ]iduringthefinal10minindicateasignificantblockageofTatmorphineincreasesin[Na ]ibylow[Na ]o,withpartial reductions in [Na ]i following exposure to Ca 2-free medium or exogenous pyruvate, but not ryanodine. Statistical significance was assessed by ANOVA followed by Bonferroni’s post hoc test; *p0.05versuscontrol,#p0.05versusTat,§p0.05versusTatmorphine;arrowsindicatetheonsetoftreatment(3independentexperiments,10–20neuronsperexperiment).TM,Tat 50 nM morphine 500 nM; low [Na ]o: 50% of the extracellular Na was substituted with Li .

Article Snippet: Pretreatments included the: AMPA receptor an- tagonist CNQX (5 M, Tocris Bioscience), opioid receptor antagonist naloxone (1.5 M, Sigma-Aldrich), -opioid receptor antagonist D-PheCys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP; 500 nM, Tocris Bioscience), -opioid receptor (DOR) antagonist naltrindole (1 M, Tocris Bioscience), -opioid receptor (KOR) antagonist nor-binaltorphimine (nor-BNI; 1 M, Tocris Bioscience), L-type calcium channel blocker nimodipine (50 M, Tocris Bioscience), endoplasmic reticulum calcium release inhibitors dantrolene (5 M, Tocris Bioscience) or ryanodine (1 M, Tocris Bioscience), and pyruvate (10 mM, Sigma-Aldrich), which is rapidly metabolized to ATP (Krisher and Prather, 2012).

Techniques:

Journal: Endocrinology

Article Title: The calcium signaling pathway regulates leydig cell steroidogenesis through a transcriptional cascade involving the nuclear receptor NR4A1 and the steroidogenic acute regulatory protein.

doi: 10.1210/en.2012-1767

Figure Lengend Snippet: FIG. 1. Blocking internal Ca2 release impairs steroidogenesis. A, Intracellular Ca2 from MA-10 Leydig cells was quantified after a 15-min treatment with vehicle (DMSO, white bar), Fsk (10 M, black bar), or Fsk plus ryanodine (Rya) (150 M, gray bar). The assay was performed in duplicate in three independent experiments. **, P 0.02. B, ELISA was used to quantify progesterone secreted by MA-10 Leydig cells after a 4-h exposure to vehicle (DMSO, white bar), Fsk (10 M, black bar), or Fsk plus ryanodine (20, 40, 75, 150 M, gray bars). Results represent data obtained from three independent experiments, each performed in duplicate. C, Progesterone levels from MA-10 Leydig cells were measured after a 4-h exposure to vehicle (DMSO, white bar), Fsk (10 M, black bar), or Fsk plus dantrolene (Dtrl) (40, 80 M, gray bars). D, Progesterone levels from MA-10 Leydig cells were measured after treatments with vehicle (DMSO, white bar), Fsk (10 M, black bars), or Fsk plus ryanodine (75 M, gray bars) for 2, 4, and 8 h as indicated. For B, C and D, results represent data obtained from three independent experiments each performed in duplicate. P 0.05 was considered statistically significant using a one-way ANOVA test. Different lowercase letters indicate a statistically significant difference.

Article Snippet: Chemicals The RyR inhibitors ryanodine and dantrolene and the CaM inhibitor W7 hydroxychloride were obtained from Tocris Bioscience (Minneapolis, MN).

Techniques: Blocking Assay, Enzyme-linked Immunosorbent Assay

Journal: Endocrinology

Article Title: The calcium signaling pathway regulates leydig cell steroidogenesis through a transcriptional cascade involving the nuclear receptor NR4A1 and the steroidogenic acute regulatory protein.

doi: 10.1210/en.2012-1767

Figure Lengend Snippet: FIG. 2. Blocking internal Ca2 release reduces Star expression in MA- 10 cells. A, Progesterone levels were measured from MA-10 Leydig cells treated for 4 h with vehicle (DMSO, white bar), OH-Chol (40 M, black bar), or OH-Chol plus ryanodine (Rya) (75 M, gray bar). B, STAR protein levels were determined by Western blot using whole-cell extracts from MA-10 Leydig cells treated for 4 h with vehicle, Fsk (10 M), or Fsk plus Rya (75 M). A representative blot from three independent experiments is shown. The bands were quantified using ImageJ software and analyzed using Student’s t test: *, P 0.05 was considered statistically significant. C, Star mRNA levels were determined by quantitative RT-PCR using total RNA isolated from MA- 10 Leydig cells treated for 2.5 h with vehicle (white bar), Fsk (10 M, black bar), or Fsk plus Rya (75 M, gray bar). Values were normalized to Rpl19 level, and results are shown as fold induction over vehicle

Article Snippet: Chemicals The RyR inhibitors ryanodine and dantrolene and the CaM inhibitor W7 hydroxychloride were obtained from Tocris Bioscience (Minneapolis, MN).

Techniques: Blocking Assay, Expressing, Western Blot, Software, Quantitative RT-PCR, Isolation

Journal: Endocrinology

Article Title: The calcium signaling pathway regulates leydig cell steroidogenesis through a transcriptional cascade involving the nuclear receptor NR4A1 and the steroidogenic acute regulatory protein.

doi: 10.1210/en.2012-1767

Figure Lengend Snippet: FIG. 3. Ca2-induced Star transcription requires the NR4A1/5A1 element. A, To locate the Ca2-responsive element within the murine Star promoter, MA-10 cells were transiently transfected with a series of 5 deletion constructs (980, 195, 144, 120, 95, and 70 bp) of the murine Star promoter and cells were treated with vehicle (white bars), 8Br-cAMP (0.5 mM, black bars), or 8Br-cAMP plus dantrolene (150 M, gray bars) for 4 h. Results from four independent experiments each in duplicate are shown as fold induction over vehicle SD. *, P 0.05; **, P 0.02. B, MA-10 cells were transiently transfected with a 980-bp mouse Star reporter, either wild type or harboring a mutation in the NR4A1/5A1 element at 100 bp. Cells were then treated with vehicle (white bars), 8Br-cAMP (0.5 mM, black bars), or 8Br-cAMP plus ryanodine (75 M, gray bars) for 4 h. Results represent data obtained from three independent experiments each performed in duplicate. P 0.05 was considered statistically significant using a one-way ANOVA test. Different lowercase letters indicate a statistically significant difference.

Article Snippet: Chemicals The RyR inhibitors ryanodine and dantrolene and the CaM inhibitor W7 hydroxychloride were obtained from Tocris Bioscience (Minneapolis, MN).

Techniques: Transfection, Construct, Mutagenesis

Journal: Endocrinology

Article Title: The calcium signaling pathway regulates leydig cell steroidogenesis through a transcriptional cascade involving the nuclear receptor NR4A1 and the steroidogenic acute regulatory protein.

doi: 10.1210/en.2012-1767

Figure Lengend Snippet: FIG. 4. NR4A1 expression is modulated by changes in internal Ca2 level. A, NR4A1 and NR5A1 protein levels were determined by Western blot using nuclear extracts from MA-10 Leydig cells treated for 1 h with vehicle, Fsk (10 M), or Fsk plus ryanodine (Rya) (75 M). A representative blot from three independent experiments is shown. Detection of lamin B was used as loading control. The bands were quantified using ImageJ software and analyzed using Student’s t test. *, P 0.05 was considered statistically significant. B, Nr4a1 mRNA level was determined by quantitative RT-PCR using RNA isolated from MA-10 Leydig cells treated for 1 h with vehicle, Fsk (10 M), or Fsk plus Rya (75 M). Values were normalized to Rpl19 level, and results are shown as fold activation over vehicle SD. *, Statistically significant difference (P 0.05). C, Transient transfections of the rat Nr4a1 promoter (747 to 50 bp) reporter were performed in MA-10 Leydig cells treated with vehicle (white bar), cAMP (0.5 mM, black bar), or cAMP plus Rya (75 M, gray bar). Results are shown as fold induction over vehicle

Article Snippet: Chemicals The RyR inhibitors ryanodine and dantrolene and the CaM inhibitor W7 hydroxychloride were obtained from Tocris Bioscience (Minneapolis, MN).

Techniques: Expressing, Western Blot, Control, Software, Quantitative RT-PCR, Isolation, Activation Assay, Transfection

Journal: The Journal of Physiology

Article Title: Dual ablation of the RyR2‐Ser2808 and RyR2‐Ser2814 sites increases propensity for pro‐arrhythmic spontaneous Ca²⁺ releases

doi: 10.1113/jp286453

Figure Lengend Snippet: Figure 1. Characterization of double knock-in mice A, generation of the double knock-in (DKI) mouse line. Please see the Methods section for a complete description. B, the total ryanodine receptor (RyR2) expression is comparable in RyR2-DKI and wild-type (WT) animals (Student’s two-sample t test, n = 6, mixed males and females). C, western blot analysis confirms the absence of phospho-epitopes Ser2808 and Ser2814 both in control conditions and in the presence of the catalytic subunit of protein kinase A (cPKA) or Ca2+/calmodulin (10 μmol/l Ca2+). A marked increase of Ser2030 phosphorylation after cPKA can be seen in the blots from both animals. D, absence of phosphorylation at Ser2808 and Ser2814 confirmed by immunofluorescence in isolated myocytes. E, echocardiographic M-mode recording in WT and RyR2-DKI mice. Ventricular mass to body weight ratio and ejection fraction were not significantly different, but the DKI mice exhibited a higher heart rate. n = 13 (5 males and 8 females) for WT; n = 13 (6 males and 7 females) for RyR2-DKI. Significant differences (†P < 0.05 vs. WT) were assessed by Student’s two-sample t test and, in the case of heart rate, by the Wilcoxon rank sum test (to test whether a data sample comes from a normal distribution, the Anderson–Darling test was used).

Article Snippet: A low dose of ryanodine (50 nmol/l; Alomone Labs, Jerusalem, Israel) was used to evoke repetitive Ca2+ sparks as previously described (Sobie et al., 2005).

Techniques: Knock-In, Expressing, Western Blot, Control, Phospho-proteomics, Immunofluorescence, Isolation

Journal: The Journal of Physiology

Article Title: Dual ablation of the RyR2‐Ser2808 and RyR2‐Ser2814 sites increases propensity for pro‐arrhythmic spontaneous Ca²⁺ releases

doi: 10.1113/jp286453

Figure Lengend Snippet: Figure 8. Spark recovery analysis indicates elevated Ca2+ sensitivity in RyR2-double knock-in cardio- myocytes A, representative line-scan image in the presence of a very low concentration of ryanodine (50 nmol/l), which can induce repetitive sparks. B, histograms of delays of Ca2+ sparks in control conditions and in the presence of 100 nmol/l isoprenaline (Iso). Black vertical lines are medians of respective spark-to-spark delay distributions. C, summary and comparison of medians with 95% confidence intervals (CIs) of each individual group. Wild type (WT): control, N = 6, n = 25, 497 spark pairs; Iso, N = 6, n = 35, 1334 spark pairs. RyR2-double knock-in (DKI): control, N = 6, n = 18, 563 spark pairs; Iso, N = 6, n = 19, 1120 spark pairs. Individual groups were tested for significance using the Kruskal–Wallis test with Dunn–Sidak post hoc test.

Article Snippet: A low dose of ryanodine (50 nmol/l; Alomone Labs, Jerusalem, Israel) was used to evoke repetitive Ca2+ sparks as previously described (Sobie et al., 2005).

Techniques: Knock-In, Concentration Assay, Control, Comparison

Journal: The EMBO Journal

Article Title: The selenocysteine-containing protein SELENOT maintains dopamine signaling in the midbrain to protect mice from hyperactivity disorder

doi: 10.1038/s44318-025-00430-3

Figure Lengend Snippet: ( A ) Schematic diagram illustrating the identification of SELENOT-interacting proteins. ( B ) Venn diagram of proteins identified by mass spectrometry of immunoprecipitants from Myc and Myc-SELT U49C . Numbers indicate number of proteins pulled down with each bait protein (Protein score > 30). ( C ) Enriched peptide numbers and protein scores for ATP2A/SERCA, IP3R, and RYR isoforms. ( D ) Clustering of enriched human phenotypes and diseases among the 242 unique SELENOT-interacting proteins. ( E , F ) Immunoprecipitation with Myc-SELT U49C to detect SERCA2, IP3R1 and RYR2. ( G – J ) IP with SERCA2 or IP3R1 to detect Myc-SELT U49C . HEK293 cells were transfected with pMyc-SELT U49C or pMyC for 48 h. Presented are the representative blots ( E , G , I ) and quantifications normalized to the non-IgG bait ( F , H , J ). n = 3 independent experiments. ( K – N ) Colocalization analyses of SERCA2 with Myc-SELT U49C and Myc-SELT. HEK293 cells were co-transfected with plasmids pFlag-SERCA and pMyc-SELT U49C or pMyc-SELT for 48 h. Presented are the representative colocalization images ( K , M ), and representative Pearson’s coefficient analysis with the calculated values ( L , N ). n = 6 cells from 3 independent experiments. Data are presented as means ± SEM and analyzed by two-tailed unpaired t -test. flag-SERCA flag-tagged SERCA, IP immunoprecipitation, IP3R inositol 1,4,5-triphosphate receptor, pFlag-SERCA2 Flag-tagged SERCA2 vector, pMyc pCMV-Myc empty vector, pMyc-SELT Myc-tagged SELENOT vector, pMyc-SELT U49C Myc-tagged SELENOT U49C vector, RYR ryanodine receptor, SERCA sarco-ER Ca 2+ ATPase. .

Article Snippet: RYR2 (rabbit) , Proteintech , 19765-1-AP.

Techniques: Mass Spectrometry, Immunoprecipitation, Transfection, Two Tailed Test, Plasmid Preparation