Journal: Scientific Reports

Article Title: Melatonin promotes triacylglycerol accumulation via MT2 receptor during differentiation in bovine intramuscular preadipocytes

doi: 10.1038/s41598-017-12780-y

Figure Lengend Snippet: Expression profiles of MT1 and MT2 at different stages of BIP differentiation. ( A ) The mRNA expression of the MT1 and MT2 genes at 3, 8 and 12 d of BIPs differentiation; ( B ) proteins level of MT1 and MT2 were quantitated using densitometry and normalized to β-actin levels; ( C ) Effect of melatonin on the mRNA and protein expression of the MT1 and MT2 genes at 8 d of BIPs differentiation. The expression of MT1 and MT2 mRNA were normalized to housekeeping genes β-actin and GAPDH. Protein level of MT1 and MT2 were quantitated using densitometry and normalized to β-actin levels. Values are presented as the means ± S.E.M. “**”represents significant differences P < 0.01. MT1 = melatonin receptors 1; MT2 = melatonin receptors 2; BIPs = bovine intramuscular preadipocytes.

Article Snippet: The membrane was treated with a blocking buffer (5% dried nonfat milk in PBS containing 0.1% Tween 20) for 1 h at room temperature and incubated overnight at 4 °C with the following antibodies: PPARγ (Cat#: bs-0530R; Bioss Inc., Woburn, MA; 1:250), MT1 (Cat#: bs-0027R; Bioss Inc., Woburn, MA; 1:250), MT2 (Cat#:bs-23279R; Bioss Inc., Woburn, MA; 1:250), HSL (Cat#:SAB4501762; Sigma, USA; 1:200), and β-actin (Cat#:bs-0061R; Bioss Inc., Woburn, MA; 1:1000).

Techniques: Expressing

Journal: Scientific Reports

Article Title: Melatonin promotes triacylglycerol accumulation via MT2 receptor during differentiation in bovine intramuscular preadipocytes

doi: 10.1038/s41598-017-12780-y

Figure Lengend Snippet: Sequences of primer pairs and amplification conditions for real time PCR.

Article Snippet: The membrane was treated with a blocking buffer (5% dried nonfat milk in PBS containing 0.1% Tween 20) for 1 h at room temperature and incubated overnight at 4 °C with the following antibodies: PPARγ (Cat#: bs-0530R; Bioss Inc., Woburn, MA; 1:250), MT1 (Cat#: bs-0027R; Bioss Inc., Woburn, MA; 1:250), MT2 (Cat#:bs-23279R; Bioss Inc., Woburn, MA; 1:250), HSL (Cat#:SAB4501762; Sigma, USA; 1:200), and β-actin (Cat#:bs-0061R; Bioss Inc., Woburn, MA; 1:1000).

Techniques: Amplification

Journal: Journal of pineal research

Article Title: Biological effects of melatonin on osteoblast/osteoclast cocultures, bone, and quality of life: Implications of a role for MT2 melatonin receptors, MEK1/2, and MEK5 in melatonin-mediated osteoblastogenesis.

doi: 10.1111/jpi.12465

Figure Lengend Snippet: FIGURE 11 Proposed mechanisms underlying melatonin’s actions on osteoblastogenesis and osteoclastogenesis. As shown, melatonin, via MT2 melatonin receptors, modulates osteoblastogenesis by coupling to both MEK1/2 and MEK5. Stimulation of MT2 melatonin receptors by melatonin in osteoblasts leads to activation of MEK5 resulting in increases in pERK5, RUNX2, NFκB, and GLUT4. MT2 melatonin receptor-mediated activation of MEK1/2 leads to increases in pERK1/2 and decreases in PPARγ, GLUT4, and IRβ. It is proposed that cross talk between MEK1/2 and MEK5 may occur where MEK5 negative regulates MEK1/2 only when both kinases are inhibited simultaneously. In MSCs, melatonin/MT2R-mediated decreases in PPARγ levels shift MSCs away from adipogenesis and toward osteogenesis. Following MSC differentiation into osteoblasts by melatonin, the mature osteoblasts begin secreting OPG to inhibit osteoclastogenesis from PBMCs

Article Snippet: For the inhibitor studies, cells were treated with 1 μmol/L of the MT2 melatonin receptor antagonist, 4P- PDOT (Tocris), 3 μmol/L of the MEK1/2 inhibitor, PD898059 (SantaCruz Biotechnology, USA), 10 μmol/L of the MEK5 inhibitor, BIX02189 (SantaCruz Biotechnology, Dallas, TX, USA), or 10 μmol/L of SC- 1- 151, a MEK1/2 and MEK5 inhibitor (generous gift from Dr. Patrick Flaherty; https://www.google.com/patents/ WO2015038743A1?cl=en) either alone or in combination with melatonin.

Techniques: Activation Assay

Journal: Journal of Pineal Research

Article Title: Overexpression of melatonin membrane receptors increases calcium‐binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

doi: 10.1111/j.1600-079x.2012.01022.x

Figure Lengend Snippet: Fig. 1. Melatonin receptor (MT1 and MT2) overexpression prevented ventral spinal cord 4.1 (VSC4.1) motoneuron death. Plasmid-mediated increase (P›) in expression was shown. Treatment groups: control cells (Con), 100 nm melatonin (24 h pretreatment), MT1-overexpressed cells, MT2-overexpressed cells, MT1 + MT2–overexpressed cells, G-protein receptor 50 (GPR50)-overexpressed cells, 25 lm L-glutamate (LGA) (24 h exposure), 100 nm melatonin (24 h pretreatment) + 25 lm LGA (24 h), MT1-overexpressed cells + 25 lm LGA (24 h), MT2-over- expressed cells + 25 lm LGA (24 h), MT1 + MT2–overexpressed cells + 25 lm LGA (24 h), GPR50-overexpressed cells + 25 lm LGA (24 h).(A) ApopTag assayshowingrepresentative imagesfromeach treatmentgroup.Arrowsindicateapoptoticcells. (B)Bargraphsindicating the percentage of apoptotic cells (ApopTag assay) and viability (trypan blue dye exclusion test) counted from each group. (C) Representative pictures show levels of MT1, MT2, GPR50, and b-actin levels (Western blotting). (D) Bar graphs indicating the changes in expression of MT1, MT2, and GPR50 over Con [Correction added on 8 Oct 2012, after first online publication: Fig. 1 Panel A has been corrected].

Article Snippet: After washing with serum-free standard medium (DMEM/F12), motoneurons were transfected in 1% low serum medium [26] over the course of 24 h at 37 C with 35 lL lipofectamine (Life Technologies, Grand Island, NY, USA) and 10 lg of GFP-tagged ORF clone of homosapiens MT1 (MTNR1A: RG210385), MT2 (MTNR1B: RG216445), or GPR50 (RG217839) plasmid (Origene, Rockville, MD, USA).

Techniques: Over Expression, Plasmid Preparation, Expressing, Control, Western Blot

Journal: Journal of Pineal Research

Article Title: Overexpression of melatonin membrane receptors increases calcium‐binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

doi: 10.1111/j.1600-079x.2012.01022.x

Figure Lengend Snippet: Fig. 2. Overexpression of MT1 and MT2 increases calbindin D28K and parvalbumin for suppressing Ca2+ rise and calpain:calpastatin ratio in ventral spinal cord 4.1 (VSC4.1) cells. Plasmid-mediated increase (P›) in expression was shown. Treatment groups: control cells (Con), 100 nm melatonin (24 h pretreatment), MT1-overexpressed cells, MT2-overexpressed cells, MT1 + MT2–overexpressed cells, G- protein receptor 50 (GPR50)-overexpressed cells, 25 lm L-glutamate (LGA) (24 h), 100 nm melatonin (24 h pretreatment) + 25 lm LGA (24 h exposure), MT1-overexpressed cells + 25 lm LGA (24 h exposure), MT2-overexpressed cells + 25 lm LGA (24 h exposure), MT1 + MT2–overexpressed cells + 25 lm LGA (24 h exposure), GPR50-overexpressed cells + 25 lm LGA (24 h exposure). (A) Determination of intracellular free Ca2+ levels at 24 h. (B) Western blot analysis to show levels of calbindin D28K, parvalbumin, calpain, calpastatin, and b-actin. (C) Bar graphs indicating the changes in expression of calbindin D28K and parvalbumin over Con. (D) Densi- tometric analysis showing the calpain:calpastatin ratio.

Article Snippet: After washing with serum-free standard medium (DMEM/F12), motoneurons were transfected in 1% low serum medium [26] over the course of 24 h at 37 C with 35 lL lipofectamine (Life Technologies, Grand Island, NY, USA) and 10 lg of GFP-tagged ORF clone of homosapiens MT1 (MTNR1A: RG210385), MT2 (MTNR1B: RG216445), or GPR50 (RG217839) plasmid (Origene, Rockville, MD, USA).

Techniques: Over Expression, Plasmid Preparation, Expressing, Control, Western Blot

Journal: Journal of Pineal Research

Article Title: Overexpression of melatonin membrane receptors increases calcium‐binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

doi: 10.1111/j.1600-079x.2012.01022.x

Figure Lengend Snippet: Fig. 3. Overexpression of MT1 and MT2 increase estrogen recep- tor (ERb:ERa) ratio and suppression inflammatory factors in ventral spinal cord 4.1 (VSC4.1) cells. Plasmid-mediated increase (P›) in expression was shown. Treatment groups: control cells (Con), 100 nm melatonin (24 h pretreatment), MT1-overexpressed cells, MT2-overexpressed cells, MT1 + MT2–overexpressed cells, G-protein receptor 50 (GPR50)-overexpressed cells, 25 lm L-glu- tamate (LGA) (24 h), 100 nm melatonin (24 h pretreat- ment) + 25 lm LGA (24 h), MT1-overexpressed cells + 25 lm LGA (24 h), MT2-overexpressed cells + 25 lm LGA (24 h), MT1 + MT2–overexpressed cells + 25 lm LGA (24 h), GPR50- overexpressed cells + 25 lm LGA (24 h). (A) Western blot anal- ysis to show levels of ERb, ERa, NF-jB, COX-2, and b-actin. (B) Densitometric analysis showing the ERb: ERa ratio. (C) Bar graphs indicating the changes in the expression of NF-jB and COX-2 over Con.

Article Snippet: After washing with serum-free standard medium (DMEM/F12), motoneurons were transfected in 1% low serum medium [26] over the course of 24 h at 37 C with 35 lL lipofectamine (Life Technologies, Grand Island, NY, USA) and 10 lg of GFP-tagged ORF clone of homosapiens MT1 (MTNR1A: RG210385), MT2 (MTNR1B: RG216445), or GPR50 (RG217839) plasmid (Origene, Rockville, MD, USA).

Techniques: Over Expression, Plasmid Preparation, Expressing, Control, Western Blot

Journal: Journal of Pineal Research

Article Title: Overexpression of melatonin membrane receptors increases calcium‐binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

doi: 10.1111/j.1600-079x.2012.01022.x

Figure Lengend Snippet: Fig. 4 Overexpression of MT1 and MT2 increases survival and angiogenesic factors in ventral spinal cord 4.1 (VSC4.1) cells. Plas- mid-mediated increase (P›) in expression was shown. Treatment groups: control cells (Con), 100 nm melatonin (24 h pretreatment), MT1-overexpressed cells, MT2-overexpressed cells, MT1 + MT2– overexpressed cells, G-protein receptor 50 (GPR50)-overexpressed cells, 25 lm L-glutamate (LGA) (24 h exposure), 100 nm melatonin (24 h pretreatment) + 25 lm LGA (24 h exposure), MT1-overex- pressed cells + 25 lm LGA (24 h), MT2-overexpressed cells + 25 lm LGA (24 h exposure), MT1 + MT2–overexpressed cells + 25 lm LGA (24 h exposure), GPR50-overexpressed cells + 25 lm LGA (24 h exposure). (A) Western blot analysis to show levels of p-Akt, Bcl-2, p-Bad, Flk-1, Flt-1, VEGF, and b-actin. (B) Bar graphs indicating the changes in expression of p-Akt, Bcl-2, and p-Bad over Con. (C) Bar graphs indicating changes in the expression of Flk-1, Flt-1, and VEGF over Con.

Article Snippet: After washing with serum-free standard medium (DMEM/F12), motoneurons were transfected in 1% low serum medium [26] over the course of 24 h at 37 C with 35 lL lipofectamine (Life Technologies, Grand Island, NY, USA) and 10 lg of GFP-tagged ORF clone of homosapiens MT1 (MTNR1A: RG210385), MT2 (MTNR1B: RG216445), or GPR50 (RG217839) plasmid (Origene, Rockville, MD, USA).

Techniques: Over Expression, Expressing, Control, Western Blot

Journal: Journal of Pineal Research

Article Title: Overexpression of melatonin membrane receptors increases calcium‐binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

doi: 10.1111/j.1600-079x.2012.01022.x

Figure Lengend Snippet: Fig. 5. Overexpression of MT1 and MT2 suppresses apoptotic pathways in ventral spinal cord 4.1 (VSC4.1) cells. Plasmid-mediated increase (P›) in expression was shown. Treatment groups: control cells (Con), 100 nm melatonin (24 h pretreatment), MT1-overexpressed cells, MT2-overexpressed cells, MT1 + MT2–overexpressed cells, G-protein receptor 50 (GPR50)-overexpressed cells, 25 lm L-glutamate (LGA) (24 h), 100 nm melatonin (24 h pretreatment) + 25 lm LGA (24 h), MT1-overexpressed cells + 25 lm LGA (24 h), MT2-over- expressed cells + 25 lm LGA (24 h), MT1 + MT2–overexpressed cells + 25 lm LGA (24 h), GPR50-overexpressed cells + 25 lm LGA (24 h). (A) Western blot analysis to show levels of Bax, Bcl-2, active caspase-9, active caspase-3, and b-actin. (B) Densitometric analysis showing the Bax:Bcl-2 ratio. (C) Bar graphs indicating the changes in expression of active caspase-9 and active caspase-3 over Con. (D) Colorimetric determination of caspase-9 and caspase-3 activities.

Article Snippet: After washing with serum-free standard medium (DMEM/F12), motoneurons were transfected in 1% low serum medium [26] over the course of 24 h at 37 C with 35 lL lipofectamine (Life Technologies, Grand Island, NY, USA) and 10 lg of GFP-tagged ORF clone of homosapiens MT1 (MTNR1A: RG210385), MT2 (MTNR1B: RG216445), or GPR50 (RG217839) plasmid (Origene, Rockville, MD, USA).

Techniques: Over Expression, Plasmid Preparation, Expressing, Control, Western Blot

Journal: Journal of Pineal Research

Article Title: Overexpression of melatonin membrane receptors increases calcium‐binding proteins and protects VSC4.1 motoneurons from glutamate toxicity through multiple mechanisms

doi: 10.1111/j.1600-079x.2012.01022.x

Figure Lengend Snippet: Fig. 6. Silencing MT1 and MT2 enhanced glutamate toxicity in ventral spinal cord 4.1 (VSC4.1) cells. We indicated the use of RNA interference (RNAi) to cause a decrease in expression. Treatment groups: control cells (Con), 25 lm L-glutamate (LGA) (24 h exposure), MT1-silenced cells + 25 lm LGA (24 h exposure); MT2-silenced cells + 25 lm LGA (24 h), MT1 + MT2-silenced cells + 25 lm LGA (24 h exposure); G-protein receptor 50 (GPR50) silenced cells + 25 lm LGA (24 h), 25 lm LGA (24 h), 25 lm LGA + 100 nm melatonin (24 h exposure), MT1-silenced cells + 25 lm LGA+ 100 nm melatonin (24h exposure), MT2 silenced cells + 25 lm LGA + 100 nm melatonin (24 h exposure), MT1 + MT2-silenced cells + 25 lm LGA + 100 nm melatonin (24 h exposure), GPR50 silenced cells + 25 lm LGA + 100 nm melatonin (24 h exposure). (A) Representative pictures showing levels of MT1, MT2, GPR50, and b-actin levels (Western blotting). (B) Bar graphs indicating the percentage of viability (trypan blue dye exclusion test) counted from each group. (C) Colorimetric determination of caspase-3 activity.

Article Snippet: After washing with serum-free standard medium (DMEM/F12), motoneurons were transfected in 1% low serum medium [26] over the course of 24 h at 37 C with 35 lL lipofectamine (Life Technologies, Grand Island, NY, USA) and 10 lg of GFP-tagged ORF clone of homosapiens MT1 (MTNR1A: RG210385), MT2 (MTNR1B: RG216445), or GPR50 (RG217839) plasmid (Origene, Rockville, MD, USA).

Techniques: Expressing, Control, Western Blot, Activity Assay

Journal: Toxicological Sciences

Article Title: Melatonin Improves Mitochondrial Function by Promoting MT1/SIRT1/PGC-1 Alpha-Dependent Mitochondrial Biogenesis in Cadmium-Induced Hepatotoxicity In Vitro

doi: 10.1093/toxsci/kfu164

Figure Lengend Snippet: Luzindole treatment abolishes the activity of the melatonin-promoted SIRT1/PGC-1 alpha signaling pathway in Cd-injured HepG2 cells. (A) The effects of melatonin and luzindole treatment on melatonin receptors (MT1 and MT2) expression, (B) SIRT1 expression, (C) SIRT1 activity, and (D) acetylated-PGC-1 alpha expression. The results were expressed as a percentage of the control, which was set at 100%. The values are presented as the mean ± SEM, ** p < 0.01 versus control group, # p < 0.05, ## p < 0.01 versus the Cd (5μM) group, and $ p < 0.05 versus the Cd (5μM) + melatonin group ( n = 4).

Article Snippet: Following protein transfer to PVDF membranes, the membranes were blocked and then incubated overnight at 4°C with antibodies against SIRT1 (1:1000, ab110304, abcam, MA), PGC-1 alpha (1:200, sc-13067, Santa Cruz, CA), acetylated-lysine (1:1000, 9441, Cell Signaling Technology), Melatonin Receptor 1A (MT1, 1:1000, ab116337, abcam, MA), p-AMPKα1/2 (1:800, sc-33524, Santa Cruz, CA), AMPKα1/2 (1:800, sc-74461, Santa Cruz, CA), Melatonin Receptor 1B (MT2, 1:1000, ab128469, abcam, MA), and β-actin (1:5000, A5441, sigma, St Louis, MO).

Techniques: Activity Assay, Expressing