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