Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Activation of ERK by anandamide in dormant blastocysts via CB1. (A) Localization of CB1 in dormant and activated blastocysts. The trophectoderm cell surface is decorated with CB1. The levels of CB1 are significantly lower in activated blastocysts than those in dormancy. (B) Rapid activation of ERK by anandamide (ANA) in blastocysts. Dormant blastocysts were cultured in vitro in the presence of 7 nM anandamide for the indicated times in minutes. Increased phosphorylation of ERK1/2(p-ERK1/2) and its translocation into nuclei were observed in dormant blastocyst trophectoderm cells within 5 min of their exposure to 7 nM anandamide, reaching a peak between 15 and 30 min. (C) Activation of ERK by anandamide is dose-dependent. Dormant blastocysts were cultured in vitro in the presence of 7 or 28 nM anandamide for 15 min. Anandamide at 7 nM activated ERK1/2 in dormant blastocyst trophectoderm cells, whereas it failed to do so at 28 nM. A CB1-selective antagonist SR141716A (SR1) at 7 nM or a MEK1/2 inhibitor U0126 at 1 μM inhibited the activation of ERK1/2 by 7 nM anandamide. (D) Total ERK remained unchanged. No changes in immunointensity for total ERK1/2 were observed in dormant blastocysts exposed to 7 nM anandamide or the vehicle. (E–G) Differential activation of ERK signaling by anandamide in CB mutant dormant blastocysts. CB1–/–, CB2–/–,or CB1–/– × CB2–/– dormant blastocysts were cultured in vitro in the presence of 7 nM anandamide. Activation of ERK1/2 in the presence of 7 nM anandamide for 15 min was abrogated by the CB1 antagonist SR141716A (SR1) in CB2–/– blastocyst, but not in CB1–/– or CB1–/– × CB2–/– blastocysts. Images shown depict TRITC-labeled antigens in red, Hoechst-labeled nuclei in blue, and the merge in pink. (Scale bars, 20 μm.)

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques: Activation Assay, Cell Culture, In Vitro, Translocation Assay, Mutagenesis, Labeling

Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Activation of ERK by anandamide in normal day-4 blastocysts via CB1. Day-4 blastocysts were cultured in vitro in the presence of 7 or 28 nM anandamide (ANA) for 15 min. Activation of ERK1/2 at lower (7 nM) but not higher (28 nM) anandamide concentration was observed. A CB1-selective antagonist SR141716A (SR1) inhibited the accumulation of phospho-ERK1/2 (p-ERK1/2) by 7 nM anandamide. Images depict TRITC-labeled antigen in red, Hoechst-labeled nuclei in blue, and the merge in pink. (Scale bar, 20 μm.) Tr, trophectoderm; ICM, inner cell mass.

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques: Activation Assay, Cell Culture, In Vitro, Concentration Assay, Labeling

Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Cannabinoid agonist CP55,940 induces activation of ERK in differentiating TS cells via CB1. (A) CB1 is expressed in TS cells. This cell line is stably transfected with the GFP gene. Images depict GFP in green, CB1 in red (TS cell surfaces), and the merge in yellow. (Scale bar, 50 μm.) (B and C) Activation of ERK in TS cells by CP55,940 (CP). TS cells were plated and expanded for 48 h. The cells were serum-starved for 5 h then exposed to different concentrations of CP for 15 min or 7 nM CP for the indicated times or to CP at 7 nM in the presence or absence of a MEK1/2 inhibitor (U0126), CB1-selective antagonist SR141716A (SR1), or CB2-selective antagonist SR144528 (SR2) for 5 min. Phosphorylation of ERK1 in differentiating TS cells was rapidly induced by 7 nM CP. U0126 or SR1, but not SR2, inhibited this activation. Quantitative analysis of ERK activation in C is expressed as percentage relative to the maximum band intensity.

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques: Activation Assay, Stable Transfection, Transfection

Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Inhibition of depolarization-induced Ca2+ influx by 28 nM anandamide in dormant blastocysts. (A) Distribution of Ca2+ channel α subunits, α1B (N-type) and α1C (L-type), in dormant blastocysts. Both inner cell mass (ICM) and trophectoderm cell (Tr) are decorated with α1B and α1C subunits shown in red, Hoechst-labeled nuclei in blue, and the merge in pink. (B) Inhibition of depolarization-induced Ca2+ influx by anandamide (ANA). Ca2+ mobilization in blastocysts was visualized with Fluo-4 acetoxymethyl ester. Depolarization-induced Ca2+ influx in dormant blastocysts after exposure to 60 mM KCl was dramatically inhibited by 28 nM anandamide but not by 7 nM. The CB1-selective antagonist SR141716A (SR1) at equimolar concentration reversed this inhibition, but the CB2-selective antagonist SR144528 (SR2) was ineffective. The relative level of intracellular Ca2+ is indicated by the fluorescent intensity, which is displayed in pseudocolor according to the color bar by using lsmib. (Scale bar, 20 μm.)

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques: Inhibition, Labeling, Concentration Assay

Journal:

Article Title: Differential G protein-coupled cannabinoid receptor signaling by anandamide directs blastocyst activation for implantation

doi: 10.1073/pnas.2436379100

Figure Lengend Snippet: Anandamide at 7 nM confers blastocyst competency to implantation via CB1

Article Snippet: Rabbit polyclonal antibodies specific to CB1 (1 μg/ml, custom made), or α 1A , α 1B , and α1C (1 μg/ml, Alomone Labs, Jerusalem), or total and phospho-ERK 1 and 2 (ERK1/2) (0.5 μg/ml, New England Biolabs) were used.

Techniques:

Journal: Nature Communications

Article Title: Impaired neuronal sodium channels cause intranodal conduction failure and reentrant arrhythmias in human sinoatrial node

doi: 10.1038/s41467-019-14039-8

Figure Lengend Snippet: Effects of Nav blockade on human SAN and atrial conduction

Article Snippet: Primary antibodies against Nav1.5 (custom-made in Dr Mohler’s lab), Nav1.6 (Alomone), Connexin-43(Cx43), Glyceraldehyde 3-phosphate dehydrogenase, and α-actinin (Sigma-Aldrich) were used to quantify corresponding proteins by western blotting and immunostaining , (Supplementary Table ).

Techniques:

Journal: Nature Communications

Article Title: Impaired neuronal sodium channels cause intranodal conduction failure and reentrant arrhythmias in human sinoatrial node

doi: 10.1038/s41467-019-14039-8

Figure Lengend Snippet: a Left to right: immunofluorescence image showing double staining of cNav1.5 (red) and Cx43 (green) in human heart 294050 cryosection with sinoatrial node (SAN) and right atrial (RA) regions ( n = 8) separated by white dotted line; magnification of image to show the distribution of cNav1.5 in the RA vs. the SAN; bar graph showing fluorescence signal intensity in the human SAN vs. RA. b Left to right: immunofluorescence image showing double staining of nNav1.6 (red) and Cx43 (green) in human heart cryosection with SAN and RA regions ( n = 7) separated by white dotted line; magnification of image to show the distribution of Nav1.6 in the RA vs. the SAN; bar graph showing fluorescence signal intensity in the human SAN vs. RA. c Left to right: cNav1.5 (red) and α-actinin (green; staining cardiomyocytes) dual staining; nNav1.6 (red) and α-actinin (green) dual staining confirm the cardiomyocyte-specific localization of cNav1.5 and nNav1.6. d Serial sections staining cNav1.5, nNav1.6, Cx43, and a nerve bundle labeled by anti-tyrosine hydroxylase (TH: staining sympathetic nerves) show that nNav1.6 and cNav1.5 are predominantly found in the myocytes relative to nerve bundles. All presented images a – d were collected from human heart 294050. e Left: representative immunoblotting bands for cNav1.5, α-actinin (marker of cardiomyocytes), and Cx43. Right: summary data of immunoblotting results of cNav1.5 protein distribution in the human SAN ( n = 6) and RA ( n = 6), compared with GAPDH (middle) and α-actinin (right), respectively. Cx43 connexin-43, GAPDH glyceraldehyde 3-phosphate dehydrogenase. Data were represented in mean ± SD. For immunostaining, analysis was done using lme4 and emmeans packages in R 3.4.4. Predictors included Heart (treated as random effect) and Condition (fixed effect). Pairwise tests between Condition levels were adjusted using Tukey’s method. Western blotting data analysis was done using two-sided t -test. Source data and uncropped versions of the western blot are provided as a file.

Article Snippet: Primary antibodies against Nav1.5 (custom-made in Dr Mohler’s lab), Nav1.6 (Alomone), Connexin-43(Cx43), Glyceraldehyde 3-phosphate dehydrogenase, and α-actinin (Sigma-Aldrich) were used to quantify corresponding proteins by western blotting and immunostaining , (Supplementary Table ).

Techniques: Immunofluorescence, Double Staining, Fluorescence, Staining, Labeling, Western Blot, Marker, Immunostaining

Journal: Nature Communications

Article Title: Impaired neuronal sodium channels cause intranodal conduction failure and reentrant arrhythmias in human sinoatrial node

doi: 10.1038/s41467-019-14039-8

Figure Lengend Snippet: a Geometry of 2D computer model. b Action potentials (AP) and their derivatives (d V /d t ) show different electrophysiological (EP) characteristics of SAN, SACP, and RA cells in the current model. Propagation of APs along the middle axis of the 2D SAN-atrium are displayed from the top to bottom with time at control conditions ( c ), Ado ( d ), and sodium current ( I Na ) 20% blockade ( e ). Blue and red numbers indicate the conduction time from SAN leading pacemaker through SAN and SACP, respectively. f Left: Ado plus I Na blockade reproduced prolonged SCL and SAN exit block. Right: representative APs and derivatives. Ado adenosine, RA right atria, SACP sinoatrial conduction pathway, SACT sinoatrial conduction time, SAN sinoatrial node.

Article Snippet: Primary antibodies against Nav1.5 (custom-made in Dr Mohler’s lab), Nav1.6 (Alomone), Connexin-43(Cx43), Glyceraldehyde 3-phosphate dehydrogenase, and α-actinin (Sigma-Aldrich) were used to quantify corresponding proteins by western blotting and immunostaining , (Supplementary Table ).

Techniques: Blocking Assay

Journal: Nature Communications

Article Title: Impaired neuronal sodium channels cause intranodal conduction failure and reentrant arrhythmias in human sinoatrial node

doi: 10.1038/s41467-019-14039-8

Figure Lengend Snippet: a Geometry of the 2D computer model with unexcitable fibrosis elements shown in light blue. b Action potentials (AP) and their derivatives (d V /d t ) show different electrophysiologic (EP) characteristics of SAN, SACP, and RA cells in the current model. Table results displaying combinations of adenosine dose and the percentage of sodium current ( I Na ) block in terms of cycle length, SACT, and threshold of SAN pacemaking and conduction impairment in control model ( c ) and HF model ( d ). Ado adenosine, HF heart failure, SACP sinoatrial conduction pathway, SACT sinoatrial conduction time, SAN, sinoatrial node, SCL sinus cycle length.

Article Snippet: Primary antibodies against Nav1.5 (custom-made in Dr Mohler’s lab), Nav1.6 (Alomone), Connexin-43(Cx43), Glyceraldehyde 3-phosphate dehydrogenase, and α-actinin (Sigma-Aldrich) were used to quantify corresponding proteins by western blotting and immunostaining , (Supplementary Table ).

Techniques: Blocking Assay