Journal: International Journal of Molecular Sciences

Article Title: TRPA1-Mediated Src Family Kinases Activity Facilitates Cortical Spreading Depression Susceptibility and Trigeminovascular System Sensitization

doi: 10.3390/ijms222212273

Figure Lengend Snippet: Saracatinib, the SFKs inhibitor, reduced the TRPA1 activator umbellulone-promoted CGRP release and IL-1β mRNA and the PKA activator dbcAMP-promoted CGRP release in the mouse TG. ( A , B ) Effects of Kreb’s solution, 40 mM KCl, 0.06% DMSO (Veh), 600 μM umbellulone (UMB) or 600 μM umbellulone + 1.5 μM saracatinib (SRCT) on CGRP release (pg/mL) and IL-1β mRNA fold change relative to β-actin in mouse TG ( n = 8 per group). ( C , D ) Correlation between CGRP release and IL-1β mRNA fold change in mouse TG treated by 0.06% DMSO, 600 μM umbellulone or 600 μM umbellulone + 1.5 μM saracatinib ( n = 8 per group). ( E , F ) Effects of Kreb’s solution, 300 μM dbcAMP or 300 μM dbcAMP + 1.5 μM saracatinib on CGRP release (pg/mL) and IL-1β mRNA fold change relative to β-actin in mouse TG ( n = 8 per group). Group data were presented as mean ± SEM. Two-tailed unpaired t-test was used for comparison between Kreb’s and KCl group, vehicle and umbellulone group, vehicle and saracatinib group in the presence of umbellulone, Kreb’s and dbcAMP group, Kreb’s and saracatinib group in the presence of dbcAMP. Two-tailed Pearson correlation was used for correlation analysis between CGRP release and IL-1β mRNA fold change in each group. Significance differences were shown as * p < 0.05, ** p < 0.01, **** p < 0.0001.

Article Snippet: The level of CGRP released into mouse TG culture media was measured by using a CGRP ELISA Kit (CSB-EQ027706MO, CUSABIO, Houston, TX, USA).

Techniques: Two Tailed Test, Comparison

Journal: International Journal of Molecular Sciences

Article Title: TRPA1-Mediated Src Family Kinases Activity Facilitates Cortical Spreading Depression Susceptibility and Trigeminovascular System Sensitization

doi: 10.3390/ijms222212273

Figure Lengend Snippet: Schematic representation of the proposed TRPA1/SFKs signaling-mediated migraine pathogenesis. TRPA1/SFKs pathway involving PKA contributes to migraine pathogenesis from multiple aspects, including cortical susceptibility to CSD, CGRP release and neuroinflammation.

Article Snippet: The level of CGRP released into mouse TG culture media was measured by using a CGRP ELISA Kit (CSB-EQ027706MO, CUSABIO, Houston, TX, USA).

Techniques:

Journal: Connective tissue research

Article Title: Crosstalk between neuropeptides SP and CGRP in regulation of BMP2-induced bone differentiation

doi: 10.1080/03008207.2017.1408604

Figure Lengend Snippet: Individual treatments of SP or CGRP did not have any direct effects but the combination of SP and CGRP partially suppressed BMP2-induced ALP activity and mineralization in C2C12 cells. A. Whole well images showing ALP staining (blue) of C2C12 cells upon treatment with SP and/or CGRP ± BMP2 (each 100 ng/ml) after 4 days in culture. B. Quantification of ALP staining using DIC images for each treatment group. Quantification was averaged from 4 independent sets of experiments, each performed in triplicates. 9 regions of interest were quantified per experiment and normalized to BMP2-only treatments. Data is reported as % of control mean value ± SEM. No significant changes were observed across different treatment groups. C. DIC images of cells with respective treatments. Scale bars measure 200 μm. D. Whole well images showing Alizarin red staining (ARS) of C2C12 cells treated with SP and/or CGRP ± BMP2 (100 ng/ml each in osteogenic differentiation medium) after 18 days. E. Quantification of ARS by solubilizing the stain according to manufacturer’s protocol. Data is reported as % of control mean value ± SEM. The neuropeptides did not have marked effects individually but the combination led to a reduction on BMP2-induced mineralization compared to other experimental groups and the controls (p<0.01). Experiments were performed in triplicates. Data is representative of 3 independent experiments. F. DIC images of cells with respective treatments. Scale bars measure 500 μm. C2C12s fused to form myotubes when there is no BMP in culture medium. BMP2 suppressed myogenic differentiation and induced mineralization.

Article Snippet: Recombinant human BMP2 (Medtronic, Minneapolis, MN), SP and CGRP (both from Bachem, Torrance, CA) were reconstituted according to manufacturer’s instructions to 1 mg/ml, aliquoted and stored at −80°C.

Techniques: Activity Assay, Staining, Control

Journal: Connective tissue research

Article Title: Crosstalk between neuropeptides SP and CGRP in regulation of BMP2-induced bone differentiation

doi: 10.1080/03008207.2017.1408604

Figure Lengend Snippet: Neither SP nor CGRP had direct effects on BMP2-induced ALP activity in MC3T3–E1 pre-osteoblasts. Individual treatments of SP and CGRP enhanced and their combination blocked BMP2-induced mineralization in these cells. A. Whole well images showing ALP staining (blue) of MC3T3–E1 pre-osteoblasts upon treatment with SP and/or CGRP ± BMP2 (each 100 ng/ml) after 4 days in culture. B. Quantification of ALP staining using DIC images for each treatment group. Quantification was averaged from 3 independent sets of experiments, each performed in triplicates. 9 regions of interest were quantified per experiment and normalized to BMP2-only treatments. Data is reported as % of control mean value ± SEM. No significant changes were observed across different treatment groups. C. DIC images of cells with respective treatments. Scale bars measure 200 μm. D. Whole well images showing Alizarin Red Staining (ARS) of MC3T3–E1 pre-osteoblasts treated with SP and/or CGRP ± BMP-2 (100 ng/ml each in osteogenic differentiation medium) after 18 days. E. Quantification of ARS by solubilizing the stain according to manufacturer’s protocol. Data is reported as % of control mean value ± SEM. SP enhanced (p<0.01) and the combination of SP and CGRP inhibited BMP2-induced mineralization compared to other experimental groups and the controls (p<0.001). Experiments were performed in triplicates. Data is representative of 3 independent experiments. F. DIC images of cells with respective treatments. Scale bars measure 200 μm.

Article Snippet: Recombinant human BMP2 (Medtronic, Minneapolis, MN), SP and CGRP (both from Bachem, Torrance, CA) were reconstituted according to manufacturer’s instructions to 1 mg/ml, aliquoted and stored at −80°C.

Techniques: Activity Assay, Staining, Control

Journal: Connective tissue research

Article Title: Crosstalk between neuropeptides SP and CGRP in regulation of BMP2-induced bone differentiation

doi: 10.1080/03008207.2017.1408604

Figure Lengend Snippet: SP enhanced BMP2 signaling and CGRP modulated its effect when delivered together. C2C12 cells were transiently transfected with pGL3-(Bre)-LUC (Firefly) and pRL-LUC (Renilla) plasmids. After 4 hours, cells were treated with respective treatments. Luciferase activity was measured within 16–20 hours. A. Dose-dependent effects of BMP2 on pGL3-(Bre)-LUC activation. Maximum response was observed at 100 ng/ml with saturation at 200ng/ml. Higher concentrations than 200 ng/ml down-regulated pGL3-(Bre)-LUC activation. B. SP and/or CGRP were added in combination with BMP2 (100ng/each) after the transfection. SP enhanced BMP2 signaling (p<0.05), while there was a clear trend for CGRP to mitigate the effect of SP (p=0.063), when delivered together. The data shown represent 6 independent experiments.

Article Snippet: Recombinant human BMP2 (Medtronic, Minneapolis, MN), SP and CGRP (both from Bachem, Torrance, CA) were reconstituted according to manufacturer’s instructions to 1 mg/ml, aliquoted and stored at −80°C.

Techniques: Transfection, Luciferase, Activity Assay, Activation Assay

Journal: Connective tissue research

Article Title: Crosstalk between neuropeptides SP and CGRP in regulation of BMP2-induced bone differentiation

doi: 10.1080/03008207.2017.1408604

Figure Lengend Snippet: Combination of SP and CGRP treatment resulted in downregulation of BMP2-induced expression ostegenic markers in C2C12 cells A. qRT-PCR for expression of osteogenic differentiation markers Alp, Col-Ia, Sp7 (Osx), Bglap (Ocn) and Runx2. Cells were treated with growth medium (GM) that contains neuropeptides SP and/or CGRP with BMP2 (each 100 ng/mL) for 2 days. Each column represents the fold change over the BMP2 control. * indicates p<0.05 and ** indicates p<0.01. Each column also shows average values of the biological replicates in each treatment. B. Gapdh was used as a reference gene since the CT value did not change across different treatment types. Combination of SP and CGRP with BMP2 induced down-regulation of Alp expression (p<0.01), while the individual treatments had no significant effect. Runx2 expression was upregulated by SP compared to BMP2-only control (p<0.05), where the combination mitigated this effect (p<0.01 compared to SP+BMP2). Therefore, there is possible convergence on regulation of Runx2 expression between the two pathways and the BMP2 signaling.

Article Snippet: Recombinant human BMP2 (Medtronic, Minneapolis, MN), SP and CGRP (both from Bachem, Torrance, CA) were reconstituted according to manufacturer’s instructions to 1 mg/ml, aliquoted and stored at −80°C.

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Connective tissue research

Article Title: Crosstalk between neuropeptides SP and CGRP in regulation of BMP2-induced bone differentiation

doi: 10.1080/03008207.2017.1408604

Figure Lengend Snippet: Hypothetical model that proposes the molecular interactions involved in the SP-CGRP-BMP2 crosstalk. Orange circles represent intermediate components of the signaling cascades. Blue circle denotes an “AND gate”, which potentially turns on a repressor of a downstream target that is required for ALP expression or mineralization. Since the combination showed down-regulation below the baseline (i.e. in comparison to BMP2-only controls), and CGRP-only had no significant effect on BMP2 signaling, we propose down-regulation of BMP2-induced osteogenic differentiation is due to an interaction between SP and CGRP. CGRP could also mitigate MAPK signaling, which suggests another possible interaction.

Article Snippet: Recombinant human BMP2 (Medtronic, Minneapolis, MN), SP and CGRP (both from Bachem, Torrance, CA) were reconstituted according to manufacturer’s instructions to 1 mg/ml, aliquoted and stored at −80°C.

Techniques: Expressing, Comparison

Journal: Frontiers in Pharmacology

Article Title: O-1602, an Agonist of Atypical Cannabinoid Receptors GPR55, Reverses the Symptoms of Depression and Detrusor Overactivity in Rats Subjected to Corticosterone Treatment

doi: 10.3389/fphar.2020.01002

Figure Lengend Snippet: Influence of the 7-day intravenous administration of O-1602 (0.25 mg/kg/day) on the levels of (A) calcitonin gene related peptide (CGRP), (B) malondialdehyde, (C) 3-nitrotyrosine, and (D) organic cation transporter 3 (OCT3) in the the bladder urothelium of rats subjected to the 14-day corticosterone treatment (20 mg/kg/day, subcutaneously). The values represent the mean + SEM (n = 15 animals per group). ***p < 0.001 versus vehicle-treated group, ^^^p < 0.001 versus corticosterone-treated group (Bonferroni’s post hoc test).

Article Snippet: The following parameters were measured in the bladder urothelium: calcitonin gene related peptide (CGRP; Biomatik, CN EKU02858), malondialdehyde (MAL; Biomatik, CN EKF57996), 3-nitrotyrosine (NIT; Lifespan Biosciences; CN LS-F40120-1), and organic cation transporter 3 (OCT3; antibodies-online, CN ABIN6227163), whereas vesicular acetylcholine transporter (VAChT; Lifespan Biosciences, CN LS-F12924-1) level was assessed in the bladder detrusor muscle.

Techniques: