Journal: Frontiers in Cellular Neuroscience

Article Title: HDAC1 Silence Promotes Neuroprotective Effects of Human Umbilical Cord-Derived Mesenchymal Stem Cells in a Mouse Model of Traumatic Brain Injury via PI3K/AKT Pathway

doi: 10.3389/fncel.2018.00498

Figure Lengend Snippet: HDAC1-silenced MSCs modulated the protein expression of PI3K/Akt pathway in hippocampus. (A) Representative Western blot bands of PTEN, phosphorylated PTEN (p-PTEN), AKT1, phosphorylated AKT1 (p-AKT1), GSK-3β, and phosphorylated GSK-3β (p-GSK-3β) in each group. (B) Quantitative protein levels were normalized with β-actin. Data are presented as mean ± SEM. * p < 0.05 vs. Vehicle, # p < 0.05 vs. MSCs.

Article Snippet: The membrane was blocked with 5% nonfat milk for 2 h at room temperature and incubated with primary antibodies respectively directed against Bcl2 (1:500, Proteintech, China), Caspase3 (1:500, Proteintech, China), Cleaved Caspase3 (1:500, Proteintech, China), HDAC1 (1:500; Abcam, Cambridge, England), PTEN (1:500; Sangon Biotech, Shanghai, China), Phospho-PTEN (Ser380/Thr382/Thr383; 1:500; Sangon Biotech, Shanghai, China), AKT-1 (1:500; Sangon Biotech, Shanghai, China), Phospho-AKT1 (Ser473; 1:500; Sangon Biotech, Shanghai, China), GSK-3β (1:500; Proteintech, Wuhan, China), Phospho-GSK-3β (Ser9; 1:500; Cell Signaling Technology, American), or β-actin (1:5,000; Sangon Biotech, Shanghai, China) at 4°C overnight.

Techniques: Expressing, Western Blot

Journal: Frontiers in Cellular Neuroscience

Article Title: HDAC1 Silence Promotes Neuroprotective Effects of Human Umbilical Cord-Derived Mesenchymal Stem Cells in a Mouse Model of Traumatic Brain Injury via PI3K/AKT Pathway

doi: 10.3389/fncel.2018.00498

Figure Lengend Snippet: LY294002 abolished the effects of HDAC1-silenced MSCs on neurologic function and the PI3K/AKT pathway of TBI mice. (A) Quantification of lesion volume. (B) Representative immunofluorescent images. (C) Quantification analysis of fluorescence intensity indicated that DCX + (red) cells in the dentate gyrus of the hippocampus in each field, Scale bar = 200 μm. (D) Representative Western blot and (E) densitometry measurement of AKT1, p-AKT1, GSK-3β, and p-GSK-3β in each group at 3 days post-TBI, (F) TST, (G) FST, (H) SPT. Data are presented as mean ± SEM. * p < 0.05 vs. Vehicle, # p < 0.05 vs. MSCs-siHDAC1.

Article Snippet: The membrane was blocked with 5% nonfat milk for 2 h at room temperature and incubated with primary antibodies respectively directed against Bcl2 (1:500, Proteintech, China), Caspase3 (1:500, Proteintech, China), Cleaved Caspase3 (1:500, Proteintech, China), HDAC1 (1:500; Abcam, Cambridge, England), PTEN (1:500; Sangon Biotech, Shanghai, China), Phospho-PTEN (Ser380/Thr382/Thr383; 1:500; Sangon Biotech, Shanghai, China), AKT-1 (1:500; Sangon Biotech, Shanghai, China), Phospho-AKT1 (Ser473; 1:500; Sangon Biotech, Shanghai, China), GSK-3β (1:500; Proteintech, Wuhan, China), Phospho-GSK-3β (Ser9; 1:500; Cell Signaling Technology, American), or β-actin (1:5,000; Sangon Biotech, Shanghai, China) at 4°C overnight.

Techniques: Fluorescence, Western Blot

Journal: Gastroenterology

Article Title: Neurotensin signaling activates microRNAs-21 and -155 and Akt, promotes tumor growth in mice, and is increased in human colon tumors.

doi: 10.1053/j.gastro.2011.07.038

Figure Lengend Snippet: Figure 4. NT regulates miR-21 and miR-155 signaling pathways in colon cancer cells. (A) Luciferase activity of the PTEN and SOCS1 3=UTRs after NT treatment (100 nmol/L) for 24 hours in HCT116 and DLD1 cells (untreated, as-miR-NC–treated, as-miR-21–treated, or as-miR-155–treated). (B) PTEN and SOCS1 mRNA levels, assessed by real-time PCR, in NT-treated (100 nmol/L, 6 h) HCT116 and DLD1 cells (untreated, as-miR-NC– treated, as-miR-21–treated, or as-miR-155–treated). (C) PTEN, SOCS1, and -actin protein levels, assessed by Western blot, in NT-treated (100 nmol/L, 6 h) HCT116 and DLD1 cells. (D) AKT phosphorylation (S473) levels, assessed by enzyme-linked immunosorbent assay, in HCT116 and DLD1 cells transfected with as-miR-NC (100 nmol/L), as-miR-21 (100 nmol/L), and/or as-miR-155 (100 nmol/L) for 24 hours and treated with NT (100 nmol/L) for 24 hours.

Article Snippet: AKT phosphorylation (S473) (DuoSet IC nzyme Linked Immunosorbent Assay, DYC887; R&D Systems, inneapolis, MN), phospho-GSK3 (S9) (DYC-1590; R&D Sysems), and phospho-p65(S536) (7834; Cell Signaling) were asessed by enzyme linked immunosorbent assay in cells transected with as-miR-21 (100 nmol/L) and/or as-miR-155 (100 mol/L) for 24 hours and treated with NT (100 nmol/L) for 24 ours.

Techniques: Protein-Protein interactions, Luciferase, Activity Assay, Real-time Polymerase Chain Reaction, Western Blot, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Transfection

Journal: Gastroenterology

Article Title: Neurotensin signaling activates microRNAs-21 and -155 and Akt, promotes tumor growth in mice, and is increased in human colon tumors.

doi: 10.1053/j.gastro.2011.07.038

Figure Lengend Snippet: Figure 5. NT activates AKT through suppression of PPP2CA by direct interaction with miR-155. (A) miR-155 binding sites in 3=UTRs of PPP2CA predicted by Lever algorithm analysis. (B) Luciferase activity of the PPP2CA 3=UTRs in HCT116 and DLD1 cells transfected with as-miR-155 (100 nmol/L) or as-miR-155 (100 nmol/L) for 24 hours. (C) Luciferase activity of the PPP2CA 3=UTRs in HCT116 and DLD1 cells treated with NT (100 nmol/L). (D) PPP2CA and -actin protein levels, assessed by Western blot, in NT-treated (100 nmol/L, 6 h) HCT116 and DLD1 cells. (E) AKT phosphorylation (S473) levels, assessed by enzyme-linked immunosorbent assay, in HCT116 and DLD1 cells transfected with miR-155 (100 nmol/L) or si-PPP2CA (100 nmol/L) for 24 hours. (F) NF-B/p65 activity, assessed by enzyme-linked immunosorbent assay, in HCT cells transfected with NT (100 nmol/L) and as-miR-NC (100 nmol/L), as-miR-21 (100 nmol/L), as-miR-155 (100 nmol/L), or with 10 nmol/L of an AKT pharmacologic inhibitor (MK-2206).

Article Snippet: AKT phosphorylation (S473) (DuoSet IC nzyme Linked Immunosorbent Assay, DYC887; R&D Systems, inneapolis, MN), phospho-GSK3 (S9) (DYC-1590; R&D Sysems), and phospho-p65(S536) (7834; Cell Signaling) were asessed by enzyme linked immunosorbent assay in cells transected with as-miR-21 (100 nmol/L) and/or as-miR-155 (100 mol/L) for 24 hours and treated with NT (100 nmol/L) for 24 ours.

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Western Blot, Phospho-proteomics, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Interferon-γ Suppresses Intestinal Epithelial Aquaporin-1 Expression via Janus Kinase and STAT3 Activation

doi: 10.1371/journal.pone.0118713

Figure Lengend Snippet:

Article Snippet: Phosphorylated AKT (Cat# AF887, R & D Systems, Minneapolis, MN) , 5% BSA in TTBS , 1:400 , goat anti-rabbit.

Techniques: Blocking Assay, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Protective Effects of Cirsilineol against Lipopolysaccharide-Induced Inflammation; Insights into HO-1, COX-2, and iNOS Modulation

doi: 10.3390/ijms24108537

Figure Lengend Snippet: CSL inhibited the activities of NF-κB and STAT-1, and upregulated the protein level of HO-1. Following LPS stimulation of HUVECs, CSL was administered at indicated concentrations or MA (20 μM) for 6 h. The protein level of HO-1 ( C ) was upregulated by CSL, and NF-κB activity ( A ) and STAT-1 phosphorylation ( B ) were suppressed. ( A ) NF-κB activity was measured in cells that were transfected with the NF-κB luciferase reporter vector, ( B ) while the phosphorylation of STAT-1 was measured by ELISA. ( C ) HO-1 expression from extracted proteins was also analyzed by ELISA. The results are presented as the mean ± SD from three independent experiments conducted in triplicate on three different days. DMSO treatment (0.2%) was used as the vehicle control, denoted as D. * p < 0.05 compared to LPS.

Article Snippet: The levels of STAT-1 phosphorylation were evaluated using ELISA kits from Abcam (ab126455, Cambridge, MA, USA).

Techniques: Activity Assay, Phospho-proteomics, Transfection, Luciferase, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Expressing, Control