Journal: Developmental Dynamics

Article Title: The endocannabinoid system regulates both ependymoglial and neuronal cell responses to a tail amputation in the axolotl

doi: 10.1002/dvdy.70035

Figure Lengend Snippet: Identification and conservation of the axolotl endocannabinoid receptors. (A) Protein sequence alignment of the putative axolotl CB1 sequence with the rat and zebrafish CB1 sequence. (B) Protein sequence alignment of the putative axolotl CB2 sequence with the rat and zebrafish CB2 sequence. Red asterisks and red boxes indicate amino acids that are conserved between all three species. (C) Western blot using the rat CB1 antibody on axolotl tail tissue demonstrates a single prominent band at ~120 kDa ( n = 3). (D) Western blot using the rat CB2 antibody on axolotl tail tissue demonstrates two bands at a similar molecular weight of ~46 kDa ( n = 3). MW = molecular weight (for each band in ladder). (E) Preadsorption control for the CB1 antibody using either CB1 or CB2 antigenic peptides ( n = 3). (F) Preadsorption control for the CB2 antibody using CB1 or CB2 antigenic peptides ( n = 3).

Article Snippet: Primary antibodies included CB1 (1:100, Alomone Labs), CB2 (1:100, Alomone Labs), GFAP (1:100, Chemicon), NeuN (1:100, Chemicon), β‐III‐tubulin (1:500, Sigma), and DCX (1:50, DSHB).

Techniques: Sequencing, Western Blot, Molecular Weight, Control

Journal: Developmental Dynamics

Article Title: The endocannabinoid system regulates both ependymoglial and neuronal cell responses to a tail amputation in the axolotl

doi: 10.1002/dvdy.70035

Figure Lengend Snippet: CB1 and CB2 are upregulated in response to tail amputation. (A) Western blot analysis demonstrates a significant upregulation of CB1 in the first 3 days after tail amputation, compared to uninjured controls ( n = 3; F (4,40) = 5.994, p = .0007, one‐way ANOVA). (B) No change in CB2 expression is shown in the first 3 days post tail amputation ( n = 3; F (4,40) = 2.779, p = .0397, one‐way ANOVA). (C) Western blot analysis demonstrates a significant upregulation of CB1 expression at both 7 and 14 days after tail amputation ( n = 3; F (2,24) = 15.97, p < .0001, one‐way ANOVA). (D) Western blot analysis demonstrates a significant upregulation of CB2 at 7 and 14 days post tail amputation ( n = 3; F (2,24) = 10.84, p = .0004, one‐way ANOVA). Uninj = uninjured tail tissue. hpa = hours post tail amputation; dpa = days post tail amputation. ns = not significant. * p < .05, ** p < .01, *** p < .001, *** *p < .0001 compared to uninjured controls. # p < .05.

Article Snippet: Primary antibodies included CB1 (1:100, Alomone Labs), CB2 (1:100, Alomone Labs), GFAP (1:100, Chemicon), NeuN (1:100, Chemicon), β‐III‐tubulin (1:500, Sigma), and DCX (1:50, DSHB).

Techniques: Western Blot, Expressing

Journal: Developmental Dynamics

Article Title: The endocannabinoid system regulates both ependymoglial and neuronal cell responses to a tail amputation in the axolotl

doi: 10.1002/dvdy.70035

Figure Lengend Snippet: CB1 and CB2 are expressed in ependymoglia and neurons in the regenerating spinal cord. (A) Schematic displays the cell‐type architecture of the axolotl spinal cord. The spinal cord is comprised of ependymoglial cells (blue) that line the central canal (cc) of the spinal cord. These ependymoglia extend GFAP + processes toward the periphery of the spinal cord. The spinal cord also contains NeuN + neurons (green) that surround the ependymoglia and extend axons that express β‐III‐tubulin. (B) Immunohistochemistry ( n = 3) shows the absence of CB1 from neuronal cell bodies (iv), and shows the co‐localization of CB1 with β‐III‐tubulin in axons (viii, yellow arrow) and with GFAP in glial cell processes (xii, blue arrow). (C) Immunohistochemistry ( n = 3) shows the absence of CB2 from neuronal cell bodies (iv), and displays the co‐localization of CB2 with β‐III‐tubulin in axons (viii, yellow arrow) and with GFAP in glial cells (xii, blue arrow). (D) Fluorescent in situ hybridization ( n = 2) demonstrates cb1 mRNA expression in both neurons (yellow arrow) and ependymoglia (blue arrow). Scale bars: 100 μm.

Article Snippet: Primary antibodies included CB1 (1:100, Alomone Labs), CB2 (1:100, Alomone Labs), GFAP (1:100, Chemicon), NeuN (1:100, Chemicon), β‐III‐tubulin (1:500, Sigma), and DCX (1:50, DSHB).

Techniques: Immunohistochemistry, In Situ Hybridization, Expressing

Journal: Developmental Dynamics

Article Title: The endocannabinoid system regulates both ependymoglial and neuronal cell responses to a tail amputation in the axolotl

doi: 10.1002/dvdy.70035

Figure Lengend Snippet: Inhibiting CB1 and CB2 receptor signaling impairs tail regeneration. (A) Representative images of tail regenerates after a 7‐day treatment with the vehicle (control, i), 1 μM AM251 (ii), or 1 μM AM630 (iii). Black dotted line indicates the original plane of amputation. Scale bar: 1 mm. (B, C) Graphs show that the proportional increase in axolotl body length was significantly reduced following either a 7‐day treatment with either 1 μM AM251 ( n = 8; B) or after a 7‐day treatment with 1 μM AM630 ( n = 8; C) compared to the vehicle control (unpaired t tests). (D) Graph shows a significant reduction in the proportional increase in axolotl body length (7 days after tail amputation) following only a 1‐day pulse treatment with either 1 μM AM251 ( n = 10) or 1 μM AM630 ( n = 10), compared to vehicle controls ( n = 10; F (2,27) = 18.86; p < .0001, one‐way ANOVA). (E) Western blot analyses show that treatment with AM251 prevented the upregulation of CB1 that normally occurs in untreated or vehicle‐treated control animals at 7‐days post tail amputation ( n = 3; Constant 7‐day bath treatment: F (3,32) = 14.69; p < .0001; 1‐day pulse treatment: F (3,32) = 18.60; p < .0001; one‐way ANOVAs). Representative blot for 1‐day pulse treatment shown. (F) Treatment with AM630 prevented the upregulation of CB2 that normally occurs in untreated or vehicle‐treated control animals at 7‐days post tail amputation ( n = 3; constant treatment: F (3,32) = 24.80; p < .0001; 1‐day pulse treatment: F (3,32) = 11.60; p < .0001; one‐way ANOVAs). Representative blot for 7‐day constant treatment shown. * *p < .01, ** *p < .001, *** *p < .0001 compared to vehicle controls. ### p < .001. #### p < .0001.

Article Snippet: Primary antibodies included CB1 (1:100, Alomone Labs), CB2 (1:100, Alomone Labs), GFAP (1:100, Chemicon), NeuN (1:100, Chemicon), β‐III‐tubulin (1:500, Sigma), and DCX (1:50, DSHB).

Techniques: Control, Western Blot

Journal: Developmental Dynamics

Article Title: The endocannabinoid system regulates both ependymoglial and neuronal cell responses to a tail amputation in the axolotl

doi: 10.1002/dvdy.70035

Figure Lengend Snippet: Inhibiting cannabinoid receptor activity reduces ependymoglial cell proliferation and upregulates GFAP + in glial cell processes. (A) Representative images of EdU + cells in the regenerating axolotl spinal cord at 7‐days post tail amputation after treatment with 1 μM AM251 (ii), 1 μM AM630 (iii), or the vehicle (control, i). White dotted circles outline the spinal cord. (B) Graph shows a significant reduction in the proportion of EdU + cells in the axolotl spinal cord at 7‐days post tail amputation after treatment with either 1 μM AM251 ( n = 4) or 1 μM AM630 ( n = 4) in comparison to vehicle controls ( n = 4; F (2,9) = 25.25; p = .0002, one‐way ANOVA). ** *p < .001 compared to vehicle controls. (C) Representative images of GFAP expression in uninjured axolotl tail tissue (i) and in regenerating tail tissue (ii) at 7‐days post tail amputation (dpa). (D) Quantified western blot data demonstrates a significant reduction in GFAP expression in the first 7‐days post tail amputation in comparison to uninjured tail tissue ( n = 3; F (3,32) = 25.97, p < .0001, one‐way ANOVA). ** *p < .001 compared to uninjured controls. (E, F) Immunohistochemistry shows GFAP expression paired with either CB1 (E) or CB2 (F) staining in the axolotl spinal cord at 7‐days post tail amputation after treatment with 1 μM AM251 (Eii), or 1 μM AM630 (Fii) or the vehicle (controls, Ei and Fi). Scale bars = 100 μm.

Article Snippet: Primary antibodies included CB1 (1:100, Alomone Labs), CB2 (1:100, Alomone Labs), GFAP (1:100, Chemicon), NeuN (1:100, Chemicon), β‐III‐tubulin (1:500, Sigma), and DCX (1:50, DSHB).

Techniques: Activity Assay, Control, Comparison, Expressing, Western Blot, Immunohistochemistry, Staining

Journal: International Journal of Molecular Sciences

Article Title: Structural Characteristics of Polysaccharide GP2a in Gardenia jasminoides and Its Immunomodulatory Effect on Macrophages

doi: 10.3390/ijms231911279

Figure Lengend Snippet: Fourier-transform infrared (FTIR) spectrum of GP2a. Bands positions are indicated in cm −1 .

Article Snippet: Thereafter, a culture medium with GP2a at various concentrations (7.5–300 µg/mL) was added and incubated for 24, 48, or 72 h. Meanwhile, untreated cells were used as blank control and the cells treated with 2 µg/mL LPS (MedChemExpress) were used as positive control.

Techniques: Fourier Transform Infrared Spectroscopy

Journal: International Journal of Molecular Sciences

Article Title: Structural Characteristics of Polysaccharide GP2a in Gardenia jasminoides and Its Immunomodulatory Effect on Macrophages

doi: 10.3390/ijms231911279

Figure Lengend Snippet: Plots of molecular weight distribution ( A ) and molecular conformation ( B ) of GP2a. ( A ) Superimposed chromatograms of GP2a obtained by size exclusion chromatography connected with multi-angle laser light scattering and refractive index (RI) detectors; the RI trace shows the signals collected by the RI detector; the molar mass trace was fitted by laser light scattering signals and RI signals, indicating molecular weight distribution. ( B ) Log–log plot of r g versus Mw; RMS—root mean square.

Article Snippet: Thereafter, a culture medium with GP2a at various concentrations (7.5–300 µg/mL) was added and incubated for 24, 48, or 72 h. Meanwhile, untreated cells were used as blank control and the cells treated with 2 µg/mL LPS (MedChemExpress) were used as positive control.

Techniques: Molecular Weight, Size-exclusion Chromatography, Refractive Index

Journal: International Journal of Molecular Sciences

Article Title: Structural Characteristics of Polysaccharide GP2a in Gardenia jasminoides and Its Immunomodulatory Effect on Macrophages

doi: 10.3390/ijms231911279

Figure Lengend Snippet: Ion chromatogram of standard monosaccharides ( A ) and GP2a hydrolysate ( B ). Peaks in ( A ): 1 is fucose (Fuc), 2 is arabinose (Ara), 3 is rhamnose (Rha), 4 is galactose (Gal), 5 is glucose (Glc), 6 is xylose (Xyl), 7 is mannose (Man), 8 is fructose (Fru), 9 is ribose (Rib), 10 is galacturonic acid (GalA), 11 is guluronic acid (GulA), 12 is glucuronic acid (GlcA), and 13 is mannuronic acid (ManA). Peaks in ( B ): 1 is Fuc, 2 is Ara, 3 is Rha, 4 is Gal, 5 is Glc, 6 is Xyl, 7 is Man, 8 is GalA, and 9 is GlcA.

Article Snippet: Thereafter, a culture medium with GP2a at various concentrations (7.5–300 µg/mL) was added and incubated for 24, 48, or 72 h. Meanwhile, untreated cells were used as blank control and the cells treated with 2 µg/mL LPS (MedChemExpress) were used as positive control.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Structural Characteristics of Polysaccharide GP2a in Gardenia jasminoides and Its Immunomodulatory Effect on Macrophages

doi: 10.3390/ijms231911279

Figure Lengend Snippet: Monosaccharide composition of GP2a.

Article Snippet: Thereafter, a culture medium with GP2a at various concentrations (7.5–300 µg/mL) was added and incubated for 24, 48, or 72 h. Meanwhile, untreated cells were used as blank control and the cells treated with 2 µg/mL LPS (MedChemExpress) were used as positive control.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Structural Characteristics of Polysaccharide GP2a in Gardenia jasminoides and Its Immunomodulatory Effect on Macrophages

doi: 10.3390/ijms231911279

Figure Lengend Snippet: Total ion chromatograms (TIC) of GP2a derivatives. ( A ) H/D, singly deuterated sample, referring to the carboxyl groups prereduced using sodium borohydride (NaBH 4 ) and secondary reduced using sodium borodeuteride (NaBD 4 ) after methylation. ( B ) D/D, doubly deuterated sample, referring to the carboxyl groups reduced using NaBD 4 before and after methylation. +EI: electron bombardment ion source.

Article Snippet: Thereafter, a culture medium with GP2a at various concentrations (7.5–300 µg/mL) was added and incubated for 24, 48, or 72 h. Meanwhile, untreated cells were used as blank control and the cells treated with 2 µg/mL LPS (MedChemExpress) were used as positive control.

Techniques: Methylation

Journal: International Journal of Molecular Sciences

Article Title: Structural Characteristics of Polysaccharide GP2a in Gardenia jasminoides and Its Immunomodulatory Effect on Macrophages

doi: 10.3390/ijms231911279

Figure Lengend Snippet: Methylation analysis result of GP2a.

Article Snippet: Thereafter, a culture medium with GP2a at various concentrations (7.5–300 µg/mL) was added and incubated for 24, 48, or 72 h. Meanwhile, untreated cells were used as blank control and the cells treated with 2 µg/mL LPS (MedChemExpress) were used as positive control.

Techniques: Methylation, Molecular Weight

Journal: International Journal of Molecular Sciences

Article Title: Structural Characteristics of Polysaccharide GP2a in Gardenia jasminoides and Its Immunomodulatory Effect on Macrophages

doi: 10.3390/ijms231911279

Figure Lengend Snippet: Scanning electron microscopy (SEM) images of GP2a at ( A ) 100×; ( B ) 250×; ( C ) 500×; ( D ) 1000×.

Article Snippet: Thereafter, a culture medium with GP2a at various concentrations (7.5–300 µg/mL) was added and incubated for 24, 48, or 72 h. Meanwhile, untreated cells were used as blank control and the cells treated with 2 µg/mL LPS (MedChemExpress) were used as positive control.

Techniques: Electron Microscopy

Journal: International Journal of Molecular Sciences

Article Title: Structural Characteristics of Polysaccharide GP2a in Gardenia jasminoides and Its Immunomodulatory Effect on Macrophages

doi: 10.3390/ijms231911279

Figure Lengend Snippet: Nuclear magnetic resonance (NMR) spectra of GP2a. ( A ) 1 H-NMR spectrum; ( B ) 13 C-NMR spectrum; ( C ) Heteronuclear single quantum relation (HSQC) spectrum; ( D ) Correlated spectroscopy (COSY) spectrum; ( E ) Heteronuclear multiple bond correlation (HMBC) spectrum; ( F ) Nuclear Overhauser effect spectroscopy (NOESY) spectrum.

Article Snippet: Thereafter, a culture medium with GP2a at various concentrations (7.5–300 µg/mL) was added and incubated for 24, 48, or 72 h. Meanwhile, untreated cells were used as blank control and the cells treated with 2 µg/mL LPS (MedChemExpress) were used as positive control.

Techniques: Nuclear Magnetic Resonance, Spectroscopy

Journal: International Journal of Molecular Sciences

Article Title: Structural Characteristics of Polysaccharide GP2a in Gardenia jasminoides and Its Immunomodulatory Effect on Macrophages

doi: 10.3390/ijms231911279

Figure Lengend Snippet: 1 H NMR and 13 C NMR chemical shifts of GP2a recorded in D 2 O.

Article Snippet: Thereafter, a culture medium with GP2a at various concentrations (7.5–300 µg/mL) was added and incubated for 24, 48, or 72 h. Meanwhile, untreated cells were used as blank control and the cells treated with 2 µg/mL LPS (MedChemExpress) were used as positive control.

Techniques: Residue

Journal: International Journal of Molecular Sciences

Article Title: Structural Characteristics of Polysaccharide GP2a in Gardenia jasminoides and Its Immunomodulatory Effect on Macrophages

doi: 10.3390/ijms231911279

Figure Lengend Snippet: Possible repeat unit structure of GP2a.

Article Snippet: Thereafter, a culture medium with GP2a at various concentrations (7.5–300 µg/mL) was added and incubated for 24, 48, or 72 h. Meanwhile, untreated cells were used as blank control and the cells treated with 2 µg/mL LPS (MedChemExpress) were used as positive control.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Structural Characteristics of Polysaccharide GP2a in Gardenia jasminoides and Its Immunomodulatory Effect on Macrophages

doi: 10.3390/ijms231911279

Figure Lengend Snippet: Cell viability of RAW 264.7 cells.

Article Snippet: Thereafter, a culture medium with GP2a at various concentrations (7.5–300 µg/mL) was added and incubated for 24, 48, or 72 h. Meanwhile, untreated cells were used as blank control and the cells treated with 2 µg/mL LPS (MedChemExpress) were used as positive control.

Techniques: Control, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Structural Characteristics of Polysaccharide GP2a in Gardenia jasminoides and Its Immunomodulatory Effect on Macrophages

doi: 10.3390/ijms231911279

Figure Lengend Snippet: Effects of GP2a on nitric oxide (NO) production in RAW 264.7 cells. Untreated cells were used as blank control; Cells treated with different GP2a concentrations (120, 240, and 300 µg/mL) were used as samples; Cells treated with lipopolysaccharide (LPS) (2 µg/mL) were used as positive control. Letter a represents significant difference compared with the blank control ( p < 0.05, p < 0.01); letter b represents significant difference compared with the positive control ( p < 0.05, p < 0.01).

Article Snippet: Thereafter, a culture medium with GP2a at various concentrations (7.5–300 µg/mL) was added and incubated for 24, 48, or 72 h. Meanwhile, untreated cells were used as blank control and the cells treated with 2 µg/mL LPS (MedChemExpress) were used as positive control.

Techniques: Control, Positive Control

Journal: International Journal of Molecular Sciences

Article Title: Structural Characteristics of Polysaccharide GP2a in Gardenia jasminoides and Its Immunomodulatory Effect on Macrophages

doi: 10.3390/ijms231911279

Figure Lengend Snippet: Effects of GP2a on cytokine production in RAW 264.7 cells. ELISA detection of tumor necrosis factor-α (TNF-α) ( A ), interferon (IFN)-γ ( B ), interleukin (IL)-1β ( C ), IL-6 ( D ), and granulocyte macrophage colony stimulating factor (GM-CSF) ( E ). Untreated cells were used as blank control; cells treated with different GP2a concentrations (120, 240, and 300 µg/mL) were used as sample; cells treated with lipopolysaccharide (LPS) (2 µg/mL) were used as positive control. Letter a represents significant difference compared with the blank control ( p < 0.05, p < 0.01); letter b represents significant difference compared with the positive control ( p < 0.05, p < 0.01).

Article Snippet: Thereafter, a culture medium with GP2a at various concentrations (7.5–300 µg/mL) was added and incubated for 24, 48, or 72 h. Meanwhile, untreated cells were used as blank control and the cells treated with 2 µg/mL LPS (MedChemExpress) were used as positive control.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Positive Control

Journal: Nanomaterials

Article Title: ZnO Nanoparticles Induce Dyslipidemia and Atherosclerotic Lesions Leading to Changes in Vascular Contractility and Cannabinoid Receptors Expression as Well as Increased Blood Pressure

doi: 10.3390/nano11092319

Figure Lengend Snippet: Effect of ZnONPs on aorta contractility. ( a ) Effect of ACPA (CB 1 receptor agonist) on contraction in aortic rings in different experimental groups. ( b ) Effect of HU308 (CB 2 receptor agonist) on contraction in aortic rings in different experimental groups. Blue bars correspond to the control non-treated group, green bars correspond to the ZnONPs treated-groups at different times; ( a ) there is a significant difference compared to the phenylephrine group with p < 0.05; ( b ) there is a significant difference compared to the control group with p < 0.05.

Article Snippet: Next, aorta sections were blocked (Animal-free blocker and diluent, Vector laboratories Inc., Burlingame, CA, USA) and incubated with rabbit polyclonal Anti-CB 1 (1:100, Alomone, Labs, Jerusalem, Israel) or rabbit polyclonal Anti-CB 2 (1:100, Alomone, Labs, Jerusalem, Israel) primary antibody and co-labeled with mouse monoclonal Anti-alpha smooth muscle Actin antibody (1:200, Abcam, Cambrige, UK) overnight.

Techniques:

Journal: Nanomaterials

Article Title: ZnO Nanoparticles Induce Dyslipidemia and Atherosclerotic Lesions Leading to Changes in Vascular Contractility and Cannabinoid Receptors Expression as Well as Increased Blood Pressure

doi: 10.3390/nano11092319

Figure Lengend Snippet: Effect of ZnONPs on the CB 1 and CB 2 receptors expression in the aorta wall. ( a ) CB 1 expression, ( b ) CB 2 expression. Blue bars correspond to the control non-treated group, green bars correspond to the ZnONPs treated-groups at different times; ( a ) there is a significant difference compared to the control group with p < 0.05.

Article Snippet: Next, aorta sections were blocked (Animal-free blocker and diluent, Vector laboratories Inc., Burlingame, CA, USA) and incubated with rabbit polyclonal Anti-CB 1 (1:100, Alomone, Labs, Jerusalem, Israel) or rabbit polyclonal Anti-CB 2 (1:100, Alomone, Labs, Jerusalem, Israel) primary antibody and co-labeled with mouse monoclonal Anti-alpha smooth muscle Actin antibody (1:200, Abcam, Cambrige, UK) overnight.

Techniques: Expressing

Journal: Nanomaterials

Article Title: ZnO Nanoparticles Induce Dyslipidemia and Atherosclerotic Lesions Leading to Changes in Vascular Contractility and Cannabinoid Receptors Expression as Well as Increased Blood Pressure

doi: 10.3390/nano11092319

Figure Lengend Snippet: Representative images showed ZnONPs effect on CB 1 and CB 2 receptors expression in aorta wall. Transmitted light images (grey). Scale bar corresponds to 50 μm. Smooth muscle α-actin was detected with a specific antibody labeled with Alexa Fluor 568 (red). CB 1 and CB 2 located on the aorta ring were detected by specific antibodies and labeled with FITC (green). Image overlap indicates a high degree of colocalization of CB 1 or CB 2, and smooth muscle α-actin (yellow). Nuclei were counterstained with DAPI dye (blue).

Article Snippet: Next, aorta sections were blocked (Animal-free blocker and diluent, Vector laboratories Inc., Burlingame, CA, USA) and incubated with rabbit polyclonal Anti-CB 1 (1:100, Alomone, Labs, Jerusalem, Israel) or rabbit polyclonal Anti-CB 2 (1:100, Alomone, Labs, Jerusalem, Israel) primary antibody and co-labeled with mouse monoclonal Anti-alpha smooth muscle Actin antibody (1:200, Abcam, Cambrige, UK) overnight.

Techniques: Expressing, Labeling

Journal: Molecular cell

Article Title: GPCRs steer G i and G s selectivity via TM5-TM6 switches as revealed by structures of serotonin receptors.

doi: 10.1016/j.molcel.2022.05.031

Figure Lengend Snippet: Figure 1. Cryo-EM structures of 5-HT4, 5-HT6, and 5-HT7 signaling complexes (A) Serotonin signaling through different subfamilies serotonin receptors. (B–E) The cryo-EM density map (left) and model (right) of the serotonin bound complexes of 5-HT4- Gs (B), 5-HT4-Gi (C), 5-HT6-Gs (D), and the 5-CT bound complex of 5-HT7-Gs (E). See also Figures S1 and S2 and Table S1.

Article Snippet: The 5-HT4 and 5-HT6 complex formation were initiated by addition of 10 mM serotonin (TargetMol), 10mg/mLNb35(Rasmussenetal., 2011b), aswell asapyrase (25mU/ml,Sigma);The5-HT7complex formationwas initiated byadditionof 10mM5-CT (Tocris), 10mg/mLNb35, aswell asapyrase (25mU/ml,Sigma).

Techniques: Cryo-EM Sample Prep

Journal: Molecular cell

Article Title: GPCRs steer G i and G s selectivity via TM5-TM6 switches as revealed by structures of serotonin receptors.

doi: 10.1016/j.molcel.2022.05.031

Figure Lengend Snippet: Figure 2. Molecular recognition of 5-HT4, 5-HT6, and 5-HT7 receptors (A–D) Conformation of the ligand-binding pocket in the serotonin-bound 5-HT4-Gs complex (A). Schematic representation of 5-HT-binding interactions. Hydrogen bonds are shown as black dashed lines. Hydrophobic contacts and amino acids are shown in green (B). Ligand-binding pocket shown as surface (C). 5-HT-induced b-arrestin2 recruitment assay for 5-HT4 mutants using NanoBiT. All data are presented as mean values ± SEM with a minimum of four technical replicates and n = 3 biological replicates. See Figure S3 for dose response curves (D). (E–H) Conformation of the ligand-binding pocket in the serotonin-bound 5-HT6-Gs complex (E). Schematic representation of 5-HT-binding interactions. Hydrogen bonds are shown as black dashed lines. Hydrophobic contacts and amino acids are shown in green (F). Ligand-binding pocket shown as surfaces (G). 5-HT- induced b-arrestin2 recruitment assay for 5-HT4 mutants using NanoBiT. All data are presented as mean values ± SEM with a minimum of four technical replicates and n = 3 biological replicates. See Figure S3 for dose response curves (H). (I–L) Conformation of the ligand-binding pocket in the 5-CT bound-5-HT7-Gs complex (I). Schematic representation of 5-HT-binding interactions. Hydrogen bonds are shown as black dashed lines. Hydrophobic contacts and amino acids are shown in green (J). Ligand-binding pocket shown as surfaces (K). Gs- cAMP accumulation results of 5-HT7 mutants activated by 5-CT. All data are presented as mean values ± SEM with a minimum of four technical replicates and n = 3 biological replicates. See Figure S3 for dose response curves (L). * The activation of the mutant is too low to determine EC50, which the corresponding DpEC50 shown as the LogEC50 value of the wild-type receptor. See also Figure S3.

Article Snippet: The 5-HT4 and 5-HT6 complex formation were initiated by addition of 10 mM serotonin (TargetMol), 10mg/mLNb35(Rasmussenetal., 2011b), aswell asapyrase (25mU/ml,Sigma);The5-HT7complex formationwas initiated byadditionof 10mM5-CT (Tocris), 10mg/mLNb35, aswell asapyrase (25mU/ml,Sigma).

Techniques: Ligand Binding Assay, Binding Assay, Activation Assay, Mutagenesis

Journal: Molecular cell

Article Title: GPCRs steer G i and G s selectivity via TM5-TM6 switches as revealed by structures of serotonin receptors.

doi: 10.1016/j.molcel.2022.05.031

Figure Lengend Snippet: Figure 3. Comparisons of serotonin binding poses and 5-CT selectivity in different serotonin receptors (A–C) Comparison of ligand recognition between 5-HT4- and 5-HT6-binding serotonin (A). Comparison of ligand recognition between 5-HT4 and 5-HT1A binding serotonin (B). Comparison of ligand recognition between 5-HT4 and 5-HT1D binding serotonin (C). (D–I) Comparison of residues in 5-HT7 recognizing 5-CT with the corresponding residues among other serotonin receptors. PDB code: 5-HT1A, 7E2Y; 5-HT1D, 7E32; 5-HT1E, 7E33; 5-HT2A, 6WHA. See also Figures S3 and S4.

Article Snippet: The 5-HT4 and 5-HT6 complex formation were initiated by addition of 10 mM serotonin (TargetMol), 10mg/mLNb35(Rasmussenetal., 2011b), aswell asapyrase (25mU/ml,Sigma);The5-HT7complex formationwas initiated byadditionof 10mM5-CT (Tocris), 10mg/mLNb35, aswell asapyrase (25mU/ml,Sigma).

Techniques: Binding Assay, Comparison

Journal: Molecular cell

Article Title: GPCRs steer G i and G s selectivity via TM5-TM6 switches as revealed by structures of serotonin receptors.

doi: 10.1016/j.molcel.2022.05.031

Figure Lengend Snippet: Figure 4. G protein coupling of 5-HT4, 5-HT6, and 5-HT7 receptors (A, D, G) Interactions between 5-HT4 and Gas subunits. Electronic interaction between the 5-HT4 and Gas protein binding interface (A). Residues model of interaction is shown in (D) and (G). (B, E, H) Interactions between 5-HT6 and Gas subunits. Electronic interaction between the 5-HT6 and Gas protein binding interface (B). Residues model of interaction is shown in (E) and (H). (C, F, I) Interactions between 5-HT7 and Gas subunits. Electronic interaction between the 5-HT7 and Gas protein binding interface (C). Residues model of interaction is shown in (F) and (I). (J) Comparison of the Ga conformation among the structures of Gs-coupled 5-HT receptors. See also Figures S4 and S6.

Article Snippet: The 5-HT4 and 5-HT6 complex formation were initiated by addition of 10 mM serotonin (TargetMol), 10mg/mLNb35(Rasmussenetal., 2011b), aswell asapyrase (25mU/ml,Sigma);The5-HT7complex formationwas initiated byadditionof 10mM5-CT (Tocris), 10mg/mLNb35, aswell asapyrase (25mU/ml,Sigma).

Techniques: Protein Binding, Comparison

Journal: Molecular cell

Article Title: GPCRs steer G i and G s selectivity via TM5-TM6 switches as revealed by structures of serotonin receptors.

doi: 10.1016/j.molcel.2022.05.031

Figure Lengend Snippet: Figure 5. Comparison of the 5-HT4-GS com- plex with the 5-HT4-Gi complex (A) Structural alignment of the 5-HT4-Gs and 5-HT4-Gi complexes. (B) Superposition of TM5 and the Gas and Gai subunits. (C) Differences in the a5 helix of Gas and Gai subunits. (D) Differences in the aN helix of Gas and Gai subunits. (E) The relative tilt/rotation angle between the C-terminal a5 helices in the Gs- and Gi-coupled complexes of 5-HT4. (F) Differences in angle and position of the whole Ga-subunit (except the N terminus helix) between the Gs- andGi/o-coupled5-HT4.See also Figures S4 and S5 and Table S4.

Article Snippet: The 5-HT4 and 5-HT6 complex formation were initiated by addition of 10 mM serotonin (TargetMol), 10mg/mLNb35(Rasmussenetal., 2011b), aswell asapyrase (25mU/ml,Sigma);The5-HT7complex formationwas initiated byadditionof 10mM5-CT (Tocris), 10mg/mLNb35, aswell asapyrase (25mU/ml,Sigma).

Techniques: Comparison

Journal: Molecular cell

Article Title: GPCRs steer G i and G s selectivity via TM5-TM6 switches as revealed by structures of serotonin receptors.

doi: 10.1016/j.molcel.2022.05.031

Figure Lengend Snippet: Figure 7. Differences between Gs- and Gi-coupled serotonin receptors (A–C) Receptor (5-HT4, A; 5-HT6, B; 5-HT7, C) interactions with the a5 terminus of Gas subunit. (D–F) Receptor (5-HT1A, D; 5-HT1D, E; 5-HT1E, F) interactions with the a5 terminus of Gai subunit. See also Figures S5 and S7 and Table S4.

Article Snippet: The 5-HT4 and 5-HT6 complex formation were initiated by addition of 10 mM serotonin (TargetMol), 10mg/mLNb35(Rasmussenetal., 2011b), aswell asapyrase (25mU/ml,Sigma);The5-HT7complex formationwas initiated byadditionof 10mM5-CT (Tocris), 10mg/mLNb35, aswell asapyrase (25mU/ml,Sigma).

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Cannabinoid receptor 2 plays a central role in renal tubular mitochondrial dysfunction and kidney ageing

doi: 10.1111/jcmm.16857

Figure Lengend Snippet: β‐catenin signalling is inhibited by CB2 gene ablation in d ‐gal‐treated mice. (A) Experimental design. Black bar indicated that mice were administered subcutaneous injections of d ‐gal at 150mg/kg/day for 6 weeks after surgery for 1 week. UNX: unilateral nephrectomy. (B) Representative micrographs showing renal expression of CB2 in different groups. Cryosections were subjected to fluorescence in situ hybridization (FISH) staining for CB2. Arrow indicates positive staining. scale bar, 25 μm. (C) Quantitative real‐time PCR results showing renal expression of CB2. * p < 0.05 versus WT mice group alone; ## p < 0.01 versus the d ‐gal‐treated WT mice group alone (n = 5–6). (D and E) Representative Western blot and quantitative data showing renal expression of β‐catenin. Numbers (1–3) indicate each individual animal in a given group. *** p < 0.001 versus WT mice group alone; ### p < 0.001 versus the d ‐gal‐treated WT mice group alone (n = 5–6). (F and G) Quantitative real‐time PCR results showing renal expression of MMP7 and AT1. * p < 0.05 and *** p < 0.001 versus WT mice group alone; # p < 0.05 and ## p < 0.01 versus the d ‐gal‐treated WT mice group alone (n = 5–6). (H) Representative micrographs showing the expression of active β‐catenin. Frozen kidney sections were stained with an antibody against active β‐catenin. Arrow indicates positive staining. Scale bar, 75μm. (I) Quantitative data showing quantification of positive staining. * p < 0.05 versus WT mice group alone; # p < 0.05 versus the d ‐gal‐treated WT mice group alone (n = 5–6)

Article Snippet: The expression of CB2 was assayed by fluorescence staining using a Fluorescence in situ hybridization kit (MK2530‐m; Boster technology).

Techniques: Expressing, Fluorescence, In Situ Hybridization, Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Cannabinoid receptor 2 plays a central role in renal tubular mitochondrial dysfunction and kidney ageing

doi: 10.1111/jcmm.16857

Figure Lengend Snippet: CB2 is upregulated in aged kidneys. (A) Representative micrographs showing CB2 expression in kidneys from 2‐month‐old and 24‐month‐old mice. Cryosections were subjected to fluorescence in situ hybridization (FISH) staining for CB2. Arrow indicates positive staining. scale bar, 25 μm. (B‐E) Representative Western blot and quantitative data showing renal expression of CB2 from 2‐month‐old and 24‐month‐old mice (B and C) or mice which were administered subcutaneous injections of d ‐gal at 150mg/kg/day for 6 weeks (D and E). Numbers (1–5) indicate each individual animal in a given group. ** p < 0.01 versus 2‐month‐old mice group or the sham control group (n = 5). (F) Representative images showing renal expression of CB2 in d ‐gal‐treated mice. Cryosections were subjected to fluorescence in situ hybridization (FISH) staining for CB2. Arrow indicates positive staining. scale bar, 25 μm. (G) Representative micrographs showing the colocalization of CB2 and various segment‐specific tubular markers in kidneys. Frozen kidney sections were stained for CB2 (red) using FISH and various segment‐specific tubular markers (green) by immunofluorescence. The following segment‐specific tubular markers were used: proximal tubule, lotus tetragonolobus lectin (LTL); distal tubule, peanut agglutinin (PNA); arrows indicate positive tubules with colocalization of CB2 and specific tubular markers. Scale bar, 25 μm. (H) Representative micrographs showing the expression of CB2 and TOMM20 in tubules in 2‐month‐old and 24‐month‐old mice. Cryosections were subjected to FISH staining of CB2 (red) and stained with TOMM20 (green) antibody by immunofluorescence. Arrows indicate positive staining. Scale bar, 25μm

Article Snippet: The expression of CB2 was assayed by fluorescence staining using a Fluorescence in situ hybridization kit (MK2530‐m; Boster technology).

Techniques: Expressing, Fluorescence, In Situ Hybridization, Staining, Western Blot, Control, Immunofluorescence

Journal: Journal of Cellular and Molecular Medicine

Article Title: Cannabinoid receptor 2 plays a central role in renal tubular mitochondrial dysfunction and kidney ageing

doi: 10.1111/jcmm.16857

Figure Lengend Snippet: CB2 gene ablation does not affect kidney ageing or mitochondrial function in young mice. (A) RT‐PCR analyses showing renal expression of CB2 in wild‐type mice (WT) and CB2 knockout mice (KO). Numbers (1–4) indicate each individual animal in a given group. (B‐F) Quantitative real‐time PCR results showing renal expression of CB2, fibronectin, α‐SMA, CollagenⅠa1 and CollagenⅢa1 in WT and KO mice. ** p < 0.01, n.s. versus WT mice group (n = 4). n.s.: no significance. (G) Representative micrographs showing Periodic acid‐Schiff (PAS) staining, Sirius red staining, senescence‐associated β‐galactosidase activity (SA‐β‐gal) staining and the expression of TOMM20. Paraffin‐embedded kidney sections were subjected to PAS and Sirius red staining. Frozen kidney sections were stained for SA‐β‐gal activity and TOMM20. Scale bar, 50 μm. (H‐K) Representative Western blot and quantitative data showing renal expression of PGC‐1α, TOMM20 and TFAM in WT and KO mice. Numbers (1–4) indicate each individual animal in a given group. n.s. versus WT mice group (n=4). n.s.: no significance. (L‐M) Quantitative real‐time PCR results showing renal expression of p16 INK4A and γH2AX in WT and KO mice. n.s. versus WT mice group (n = 4). n.s.: no significance. (N‐R) Representative Western blot and quantitative data showing renal expression of β‐catenin, MMP7, Snail1 and AT1 in WT and KO mice. Numbers (1–4) indicate each individual animal in a given group. n.s. versus WT mice group (n = 4). n.s.: no significance

Article Snippet: The expression of CB2 was assayed by fluorescence staining using a Fluorescence in situ hybridization kit (MK2530‐m; Boster technology).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Knock-Out, Real-time Polymerase Chain Reaction, Staining, Activity Assay, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Cannabinoid receptor 2 plays a central role in renal tubular mitochondrial dysfunction and kidney ageing

doi: 10.1111/jcmm.16857

Figure Lengend Snippet: CB2 deficiency protects renal mitochondrial homeostasis in the accelerated ageing mice. (A) Representative micrographs showing renal expression of PGC‐1α and TOMM20 in different groups. Paraffin‐embedded kidney sections were immunostained with an antibody against PGC‐1α or TOMM20. Arrows indicate positive staining. Scale bar, 50 μm. (B‐C) Quantitative data showing quantification of positive staining. * p < 0.05, *** p < 0.001 versus WT mice group alone; # p < 0.05, ### p < 0.001 versus the d ‐gal‐treated WT mice group alone (n = 5–6). (D) Representative graph showing the production of adenosine triphosphate (ATP) in different groups. * p < 0.05 versus WT mice group alone; ## p < 0.01 versus the d ‐gal‐treated WT mice group alone (n = 5–6). (E–H) Representative Western blot and quantitative data showing renal expression of PGC‐1α, TOMM20 and Cytb. Numbers (1–3) indicate each individual animal in a given group. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the WT mice group alone; # p < 0.05, ### p < 0.001 versus the d ‐gal‐treated WT mice group alone (n = 5–6). (I) Representative transmission electron microscopy graphs showing mitochondrial ultrastructure of renal tubular cells in different groups. Arrows indicate damaged and abnormal‐shaped mitochondria. Scale bar, 1μm

Article Snippet: The expression of CB2 was assayed by fluorescence staining using a Fluorescence in situ hybridization kit (MK2530‐m; Boster technology).

Techniques: Expressing, Staining, Western Blot, Transmission Assay, Electron Microscopy

Journal: Journal of Cellular and Molecular Medicine

Article Title: Cannabinoid receptor 2 plays a central role in renal tubular mitochondrial dysfunction and kidney ageing

doi: 10.1111/jcmm.16857

Figure Lengend Snippet: CB2 gene ablation ameliorates kidney ageing. (A) Representative micrographs showing renal expression of γH2AX and SA‐β‐gal activity in different groups. Paraffin‐embedded kidney sections were immunostained with an antibody against γH2AX (top). Frozen kidney sections were stained for SA‐β‐gal activity (bottom). Arrows indicate positive staining. Scale bar, 50 μm. (B‐C) Quantitative data showing quantification of positive staining. ** p < 0.01 versus WT mice group alone; ## p < 0.01 versus the d ‐gal‐treated WT mice group alone (n = 5–6). (D–G) Representative Western blot and quantitative data showing renal expression of p16 INK4A , γH2AX and p19 ARF in different groups. Numbers (1–3) indicate each individual animal in a given group. ** p < 0.01, *** p < 0.001 versus the WT mice group alone; # p < 0.05, ## p < 0.01, ### p < 0.001 versus the d ‐gal‐treated WT mice group alone (n = 5–6). (H and I) Representative micrographs showing renal expression of klotho in different groups (H). Paraffin‐embedded kidney sections were immunostained with an antibody against klotho. Arrows indicate positive staining. Scale bar, 50 μm. (I) Quantitative data showing quantification of positive staining. *** p < 0.001 versus the WT mice group alone; ### p < 0.001 versus the d ‐gal‐treated WT mice group alone (n = 5–6)

Article Snippet: The expression of CB2 was assayed by fluorescence staining using a Fluorescence in situ hybridization kit (MK2530‐m; Boster technology).

Techniques: Expressing, Activity Assay, Staining, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Cannabinoid receptor 2 plays a central role in renal tubular mitochondrial dysfunction and kidney ageing

doi: 10.1111/jcmm.16857

Figure Lengend Snippet: CB2 deficiency retards age‐related kidney fibrosis. (A and B) Quantitative data showing serum creatinine (Scr) and blood urea nitrogen (BUN) levels in different groups. n.s.: no significance. (C–E) Representative Western blot and quantitative data showing renal expression of fibronectin and α‐SMA in different groups. Numbers (1–3) indicate each individual animal in a given group. * p < 0.05 versus the WT mice group alone; # p < 0.05, ## p < 0.01 versus the d ‐gal‐treated WT mice group alone (n = 5–6). (F–H) Representative micrographs showing renal expression of fibronectin and Sirius red staining in different groups. Paraffin‐embedded kidney sections were stained with Sirius red and were immunostained with an antibody against fibronectin. Arrows indicate positive staining. Scale bar, 50 μm. Quantitative data showing quantification of positive staining of fibronectin (G) and fibrotic area (H). ** p < 0.01, *** p < 0.001 versus the WT mice group alone; ## p < 0.01, ### p < 0.001versus the d ‐gal‐treated WT mice group alone (n = 5–6)

Article Snippet: The expression of CB2 was assayed by fluorescence staining using a Fluorescence in situ hybridization kit (MK2530‐m; Boster technology).

Techniques: Western Blot, Expressing, Staining

Journal: Journal of Cellular and Molecular Medicine

Article Title: Cannabinoid receptor 2 plays a central role in renal tubular mitochondrial dysfunction and kidney ageing

doi: 10.1111/jcmm.16857

Figure Lengend Snippet: CB2 induces mitochondrial dysfunction and cellular senescence in vitro. (A–I) Representative Western blot and quantitative data showing the expression of CB2, PGC‐1α, Cytb, TOMM20, COX1, COX2, p16 INK4A , γH2AX in HKC‐8 cells. HKC‐8 cells were transfected with CB2 expression plasmid (pCMV‐CB2) for 24 h. * p < 0.05, ** p < 0.01 versus the pcDNA3 group (n = 3). (J–T) Representative Western blot and quantitative data showing the expression of CB2, PGC‐1α, Cytb, TOMM20, COX1, COX2, TFAM, p16 INK4A , γH2AX and p14 ARF in HKC‐8 cells. HKC‐8 cells were treated with AM1241 (10 μM) for 48h. * p < 0.05, ** p < 0.01 versus the control group (n = 3)

Article Snippet: The expression of CB2 was assayed by fluorescence staining using a Fluorescence in situ hybridization kit (MK2530‐m; Boster technology).

Techniques: In Vitro, Western Blot, Expressing, Transfection, Plasmid Preparation, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Cannabinoid receptor 2 plays a central role in renal tubular mitochondrial dysfunction and kidney ageing

doi: 10.1111/jcmm.16857

Figure Lengend Snippet: β‐catenin plays a mediative role in CB2‐induced mitochondrial dysfunction and cellular senescence. (A–E) Representative Western blot (A, F) and quantitative data (C–E, G and H) showing the expression of COX1, TFAM, TOMM20, p14 ARF and γH2AX in HKC‐8 cells. HKC‐8 cells were treated with AM1241 (10 μM) for 48 h and pretreated with XL‐001 (10 μM) for 1 h. Quantitative data graph (B) showing the production of adenosine triphosphate (ATP) in HKC‐8 cells. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the control group alone; # p < 0.05, ## p < 0.01, ### p < 0.001versus the AM1241 group alone (n = 3). (I–N) Representative Western blot (I, M) and quantitative data (J–L, N) showing the expression of PGC‐1α, Cytb, TOMM20 and p16 INK4A in HKC‐8 cells. HKC‐8 cells were transfected with CB2 expression plasmid (pCMV‐CB2), followed by the stimulation of ICG‐001 at 10μM for 24 h * p < 0.05, ** p < 0.01, *** p < 0.001 versus the control group alone; # p < 0.05, ## p < 0.01versus the pCMV‐CB2 group alone (n = 3)

Article Snippet: The expression of CB2 was assayed by fluorescence staining using a Fluorescence in situ hybridization kit (MK2530‐m; Boster technology).

Techniques: Western Blot, Expressing, Control, Transfection, Plasmid Preparation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Cannabinoid receptor 2 plays a central role in renal tubular mitochondrial dysfunction and kidney ageing

doi: 10.1111/jcmm.16857

Figure Lengend Snippet: CB2 plays a central role in the accelerated ageing in renal tubular cells. (A–H) Representative Western blot and quantitative data showing the expression of CB2, PGC‐1α, TOMM20, COX1, p16 INK4A , p14 ARF and β‐catenin in HKC‐8 cells. HKC‐8 cells were treated with D‐gal at 10mg/ml for 72h and pretreated with XL‐001 (10μM) for 1 h. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the control group alone; # p < 0.05, ## p < 0.01, ### p < 0.001 versus the d ‐gal group alone (n = 3). (I and J) Representative micrographs and quantitative data showing SA‐β‐gal activity in different groups. Frozen kidney sections were stained for SA‐β‐gal activity. Arrows indicate positive staining. Scale bar, 20 μm. *** p < 0.001 versus the control group alone; ### p < 0.001 versus the d ‐gal group alone (n = 3). (K–N) Representative Western blot and quantitative data showing renal expression of PGC‐1α, TOMM20 and p14 ARF in HKC‐8 cells. HKC‐8 cells were treated with D‐gal at 10mg/ml or cotreated with AM1241 (10 μM) for 72 h and pretreated with ICG‐001 (10 μM) for 1 h. * p < 0.05, ** p < 0.01, *** p < 0.001 versus the control group alone; # p < 0.05, ## p < 0.01, ### p < 0.001 versus the d ‐gal group alone; †† p < 0.01, ††† p < 0.001 versus the d ‐gal+AM1241 group alone (n = 3). (O and P) Representative micrographs and quantitative data showing SA‐β‐gal activity in different groups. Frozen kidney sections were stained for SA‐β‐gal activity. Arrows indicate positive staining. Scale bar, 20 μm. ** p < 0.01 versus the control group alone; ### p < 0.001 versus the d ‐gal group alone; ††† p < 0.001 versus the d ‐gal+AM1241 group alone (n = 3)

Article Snippet: The expression of CB2 was assayed by fluorescence staining using a Fluorescence in situ hybridization kit (MK2530‐m; Boster technology).

Techniques: Western Blot, Expressing, Control, Activity Assay, Staining