Journal: Aging (Albany NY)

Article Title: Neuroprotective and neurogenic effects of novel tetramethylpyrazine derivative T-006 in Parkinson’s disease models through activating the MEF2-PGC1α and BDNF/CREB pathways

doi: 10.18632/aging.103551

Figure Lengend Snippet: T-006 stimulates MEF2D/PGC1α/Nrf2 signal pathway through regulation of the Akt/GSK3β pathway in PD animal models. ( A ) Representative Western blots and densitometric analysis the expression of CDK5, p-MEF2D, MEF2D and PGC1α. ( B , E ) Representative Western blots and densitometric analysis of the expression of p-Akt and p-GSK3β. ( C , F ) Representative Western blots and densitometric analysis of the expression of Nrf2, HO-1 and TFAM. ( D ) Representative Western blots and densitometric analysis the expression of MEF2D and PGC1α. Data are expressed as mean±SEM (n=3 per group). # P<0.05, ## P<0.01, ### P<0.001 vs. sham group, and * P<0.05, ** P<0.01 and *** P<0.001vs. MPTP or 6-OHDA group.

Article Snippet: After blocking with a blocking buffer, the polyvinyldifluoride membranes were co-incubated using primary antibodies overnight at 4 o C respectively against TH (Cell Signaling Technology), CDK5, phospho-Ser444-MEF2D (p-MEF2D, Merck Millipore, Germany), MEF2D (Merck Millipore, Germany), PGC1α, Nrf2, HO-1, TFAM, phospho-Ser473-Akt (p-Akt), total-Akt (Akt), p-GSK3β, total-GSK3β (GSK3β), phospho-Ser133-CREB (p-CREB, Cell Signaling Technology), CREB (Cell Signaling Technology), mature BDNF (abcam, Britain), synaptophysin (SYP, Cell Signaling Technology) and β-actin.

Techniques: Western Blot, Expressing

Journal: Aging (Albany NY)

Article Title: Neuroprotective and neurogenic effects of novel tetramethylpyrazine derivative T-006 in Parkinson’s disease models through activating the MEF2-PGC1α and BDNF/CREB pathways

doi: 10.18632/aging.103551

Figure Lengend Snippet: T-006 prevents the loss of SN DA neuron loss by activating MEF2D/PGC1α/GSK3β signal pathway. Representative images of middle brain sections co-stained with antibodies against ( A , D ) TH (green) and p-GSK3β (red); ( B , E ) TH (red) and MEF2D (green); ( C , F ) TH (green) and PGC1α (red). DAPI (blue) indicates nucleus. ( G , H ) Quantitative analysis of immunofluorescence intensity in TH-positive cells. Data are expressed as mean±SEM (n=3 to 4 per group). # P<0.05, ### P<0.001 vs. sham group, and * P<0.05, ** P<0.01 vs. MPP + or 6-OHDA group.

Article Snippet: After blocking with a blocking buffer, the polyvinyldifluoride membranes were co-incubated using primary antibodies overnight at 4 o C respectively against TH (Cell Signaling Technology), CDK5, phospho-Ser444-MEF2D (p-MEF2D, Merck Millipore, Germany), MEF2D (Merck Millipore, Germany), PGC1α, Nrf2, HO-1, TFAM, phospho-Ser473-Akt (p-Akt), total-Akt (Akt), p-GSK3β, total-GSK3β (GSK3β), phospho-Ser133-CREB (p-CREB, Cell Signaling Technology), CREB (Cell Signaling Technology), mature BDNF (abcam, Britain), synaptophysin (SYP, Cell Signaling Technology) and β-actin.

Techniques: Staining, Immunofluorescence

Journal: Aging (Albany NY)

Article Title: Neuroprotective and neurogenic effects of novel tetramethylpyrazine derivative T-006 in Parkinson’s disease models through activating the MEF2-PGC1α and BDNF/CREB pathways

doi: 10.18632/aging.103551

Figure Lengend Snippet: T-006 activates the MEF2/PGC1α/Nrf2 pathway through regulation of the Akt-GSK3β pathway. A53T and corrected DA neurons treated with T-006 and the positive drug at the indicated concentration for 24 hours. For MPP + -treatment assay, CGNs were pretreated with or without LY294002 (1 μM), Akt-iv (1 μM), or LiCl (10 μM) for 2 h, incubated with or without T-006 for 2 h, and finally exposed to MPP + . Cell viability was examined using an MTT assay. Luciferase reporter gene assays respectively included MEF2 ( A ), PGC1α ( B ), PGC1α-ΔMEF2 ( C ) and ARE ( D ). ( E ) Representative images of neurons co-stained with antibody against Nrf2 (green). DAPI (blue) indicates nucleus. ( F ) Luciferase reporter gene assays of MEF2 with MPP + induction. ( G – K ) respectively represent the fold changes of CDK5, MEF2D, PGC1α, Nrf1 and Nrf2 at mRNA level. ( L ) Effect of MEF2D reduction on MPP + -induced neurotoxicity in CGNs. ( M ) Effects of Akt pathway inhibitor LY294002 and GSK3β inhibitor on MEF2 transcriptional activity. ( N ) Effects of Akt pathway inhibitors LY294002 and Akt-iv, and GSK3β inhibitor on MPP + -induced neurotoxicity in CGNs. Data above are all from three independent experiments, expressed as mean±SEM. # P<0.05, ## P<0.01 and ### P<0.001 vs. control (Ctrl) group, and * P<0.05, ** P<0.01, *** P<0.001 vs. MPP + group.

Article Snippet: After blocking with a blocking buffer, the polyvinyldifluoride membranes were co-incubated using primary antibodies overnight at 4 o C respectively against TH (Cell Signaling Technology), CDK5, phospho-Ser444-MEF2D (p-MEF2D, Merck Millipore, Germany), MEF2D (Merck Millipore, Germany), PGC1α, Nrf2, HO-1, TFAM, phospho-Ser473-Akt (p-Akt), total-Akt (Akt), p-GSK3β, total-GSK3β (GSK3β), phospho-Ser133-CREB (p-CREB, Cell Signaling Technology), CREB (Cell Signaling Technology), mature BDNF (abcam, Britain), synaptophysin (SYP, Cell Signaling Technology) and β-actin.

Techniques: Concentration Assay, Incubation, MTT Assay, Luciferase, Staining, Activity Assay, Control

Journal: Oncogene

Article Title: Divergent functions for eIF4E and S6 kinase by Sonic hedgehog mitogenic signaling in the developing cerebellum

doi: 10.1038/onc.2010.564

Figure Lengend Snippet: Shh-treated proliferating CGNPs show increased levels of translation initiation components and suppressed S6 kinase activity. (A) Western blot analysis of protein isolated from vehicle- or Shh-treated CGNP primary cultures. Shh treatment stimulates mTOR activity as demonstrated by eIF4G and 4EBP2 hyperphosphorylation but blocks S6 kinase activity measured by rpS6 phosphorylation. Levels of the mTOR effector eIF4E increase in Shh-treated CGNPs. (B) Quantification of western blots by densitometry. Phosphorylated protein levels were compared to total protein levels after normalization to β-Tubulin n=3, (**p<0.002, ***p<0.0005). (C to I) Immunofluorescence analysis of vehicle- and Shh-treated CGNPs plated on coverslips. eIF4E increases in Shh-treated CGNPs (D compared to C) while phospho-S235/236-rpS6 accumulates in vehicle-treated CGNPs (E compared to F). Total rpS6 levels do not change in proliferating or differentiated cells (G, H). Furthermore phospho-S235/236-rpS6 is expressed in vehicle-treated CGNPs marked by the differentiation marker, MEF2D (I arrowheads), while eIF4E protein colocalizes with PCNA positive cells (J arrowheads) in Shh-treated CGNPs. (K) Quantification of immunostaining in C-H, n=3, (***p<0.0001, **p<0.001). (L) Percentage of cells expressing eIF4E only, PCNA only or both eIF4E and PCNA in Shh-treated CGNPs. (M) Percentage of cells expressing MEF2D only, phospho-rps6 only or both MEF2D and phospho-rpS6 in vehicle-treated CGNPs.

Article Snippet: Primary antibodies included eIF4E, phospho-S6 (235/236), GFAP, NeuN (Cell Signaling), MEF2D, p27 (BD-PharMingen), BrdU (Becton Dickinson), and PCNA (Calbiochem).

Techniques: Activity Assay, Western Blot, Isolation, Phospho-proteomics, Immunofluorescence, Marker, Immunostaining, Expressing

Journal: Oncology Letters

Article Title: Absent expression of miR-30a promotes the growth of lung cancer cells by targeting MEF2D

doi: 10.3892/ol.2018.8719

Figure Lengend Snippet: MEF2D is expressed at a high level in lung cancer tissue samples. (A) Immunohistochemical analysis of the expression of MEF2D was performed in tumor and paired non-cancerous tissues. (B) Western blot analysis of the protein expression of MEF2D in lung cancer and paired non-cancerous tissue. Quantification of western blots is shown below corresponding bands. (C) Reverse transcription-quantitative polymerase chain reaction analysis of mRNA levels of MEF2D in paired patient cancer and normal tissues. MEF2D, myocyte enhancer factor 2D; T, tumor; N, non-cancerous.

Article Snippet: Rabbit anti-human MEF2D antibody (cat. no. HPA004807; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used to detect the expression of MEF2D.

Techniques: Immunohistochemical staining, Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: Oncology Letters

Article Title: Absent expression of miR-30a promotes the growth of lung cancer cells by targeting MEF2D

doi: 10.3892/ol.2018.8719

Figure Lengend Snippet: miR-30a is underexpressed in lung cancer samples. (A) Reverse transcription-quantitative polymerase chain reaction analysis of the expression of miR-30a was performed in tumor tissues and paired non-cancerous tissues. (B) Correlation curve between mRNA expression level of MEF2D and expression level of miR-30a. MEF2D, myocyte enhancer factor 2D; miR, microRNA.

Article Snippet: Rabbit anti-human MEF2D antibody (cat. no. HPA004807; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used to detect the expression of MEF2D.

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: Oncology Letters

Article Title: Absent expression of miR-30a promotes the growth of lung cancer cells by targeting MEF2D

doi: 10.3892/ol.2018.8719

Figure Lengend Snippet: miR-30a regulates the expression of MEF2D in lung cancer. (A) An miR-30a binding site was predicted to exist in the 3′UTR of MEF2D mRNA. (B) miR-30a mimics and a luciferase vector with the MEF2D 3′UTR were co-transfected into A549 cells and the luciferase activity was detected. (C) Western blot analysis of the protein expression of MEF2D was performed in A549 cells following transfection with miR-30a mimics and miR-NC. Quantified results of the western blot analysis are shown below the corresponding bands. MEF2D, myocyte enhancer factor 2D; miR, microRNA; NC, negative control; 3′UTR, 3′untranslated region. *P<0.05 vs. NC.

Article Snippet: Rabbit anti-human MEF2D antibody (cat. no. HPA004807; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used to detect the expression of MEF2D.

Techniques: Expressing, Binding Assay, Luciferase, Plasmid Preparation, Transfection, Activity Assay, Western Blot, Negative Control

Journal: Oncology Letters

Article Title: Absent expression of miR-30a promotes the growth of lung cancer cells by targeting MEF2D

doi: 10.3892/ol.2018.8719

Figure Lengend Snippet: miR-30a suppresses the proliferation and colony formation of A549 cells. (A) Growth rates of A549 cells transfected with miR-30a mimics were determined. (B) Numbers of colonies and (C) percentage area of formed colonies were measured in the cells transfected with miR-30a mimics. MEF2D, myocyte enhancer factor 2D; miR, microRNA; NC, negative control. *P<0.05 vs. NC.

Article Snippet: Rabbit anti-human MEF2D antibody (cat. no. HPA004807; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used to detect the expression of MEF2D.

Techniques: Transfection, Negative Control

Journal: Oncology Letters

Article Title: Absent expression of miR-30a promotes the growth of lung cancer cells by targeting MEF2D

doi: 10.3892/ol.2018.8719

Figure Lengend Snippet: miR-30a promotes the apoptosis of A549 cells. (A) Annexin V/PI staining analysis was performed on the A549 cells using FACS. (B) Western blot analysis of cleaved caspase-3 was performed in the miR-30a-mimics-transfected A549 cells. Quantified results of the western blot analysis are shown below the corresponding bands. MEF2D, myocyte enhancer factor 2D; miR, microRNA; NC, negative control; PI, propidium iodide.

Article Snippet: Rabbit anti-human MEF2D antibody (cat. no. HPA004807; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) was used to detect the expression of MEF2D.

Techniques: Staining, Western Blot, Transfection, Negative Control