Journal: Journal of Cell Science

Article Title: Presenilin-1 acts via Id1 to regulate the function of muscle satellite cells in a ?-secretase-independent manner

doi: 10.1242/jcs.049742

Figure Lengend Snippet: PS1 negatively regulates MyoD expression in a γ-secretase-independent mechanism. In order to obtain sufficient material for biochemical analysis, we also used the adult mouse muscle-derived C2C12 myogenic cell line. (A) Immunoblot analysis showed that proliferating C2C12 cells expressed PS1, and that the siRNA-targeting PS1 (PS1si-1 and PS1si-2) was effective at reducing PS1 protein levels. (B) As with primary myoblasts, exposure of C2C12 myoblasts to siRNA against PS1 promoted myogenic differentiation, as shown by immunostaining for MyHC, in both growth (GM) and differentiation medium (DM) (quantified in C for DM). Importantly, treatment with DAPT in GM for 3 days to inhibit γ-secretase activity did not reproduce the effects of PS1 knockdown, being unable to induce expression of MyHC in C2C12 cells. (D) Immunblot analysis of siRNA-transfected C2C12 cells cultured in GM or DM for 3 days after transfection confirmed increased MyHC expression. Importantly, Pax7 levels were significantly reduced by PS1 knockdown under both culture conditions. (E-I) PS1 was also constitutively expressed in C2C12 myoblasts by transient transfection with PS1-expression vector (pMSCV-PS1-IRES-GFP). (F) Immunocytochemical analysis for MyHC revealed that myogenic differentiation was again inhibited. (G) Immunoblot analysis of transfected C2C12 cells constitutively expressing PS1, illustrating that inhibiting γ-secretase activity by exposure to 1 μM DAPT for 2.5 days did not alter the marked reduction in MyHC levels (quantified in H). (I) Immunoblot analysis demonstrated that constitutive PS1 expression in C2C12 cells cultured in GM for 24 hours after transfection did not affect the levels of Notch1 ICD, Notch2 ICD or Hes1, but that MyoD was significantly reduced, compared to the tubulin protein loading control. (J) Similar results were obtained using a V5-tagged PS1 expression vector transfected into C2C12 cells. Immunostaining showed that MyoD expression was reduced to a similar degree, with or without exposure to 1 μM DAPT to inhibit γ-secretase activity (quantified in K). Data from at least three independent experiments is shown ± s.d. Asterisks in C, H and K indicate that data are significantly different from control values (P<0.05). Scale bars: 100 μm (B), 20 μm (E), 30 μm (F) and 10 μm (J).

Article Snippet: Antibodies were obtained from the following sources: mouse and rabbit anti-PS1 antibodies from Millipore (Bedford, MA); mouse anti-GFP from Roche (Basel, Switzerland); rabbit anti-PS2 antibody from Abcam; mouse anti-Notch1 antibody from BD Biosciences; rat anti-Ki67 from DAKO; goat anti-PS1 antibody, rabbit anti-Id1 antibody, rabbit anti-Hes1 antibody goat anti-Notch2 antibody and rabbit anti-MyoD antibody were obtained from Santa Cruz (Santa Cruz, CA); mouse anti-developmental myosin heavy chain (dMyHC) antibody from Novocastra (Newcastle, UK); mouse anti-MyHC (MF20), anti-Pax7 antibody, anti-Myog antibody (F5D) and anti-tubulin antibody (E7) were obtained from the DSHB (Iowa City, IA); rabbit anti-p-histone H3 antibody from Cell Signaling Technology (Beverly, MA) and Topro-3, rabbit anti-GFP antibody and mouse anti-V5 antibody from Invitrogen (Carlsbad, CA).

Techniques: Expressing, Derivative Assay, Western Blot, Immunostaining, Activity Assay, Knockdown, Transfection, Cell Culture, Plasmid Preparation, Control

Journal: Oncology Letters

Article Title: miR-29a activates Hes1 by targeting Nfia in esophageal carcinoma cell line TE-1

doi: 10.3892/ol.2014.2678

Figure Lengend Snippet: Overexpression of miR-29a upregulates Hes1 and downregulates Nfia. (A) Notch1, Hes1, and Nfia mRNA expression levels were detected using reverse transcription-quantitative polymerase chain reaction in TE-1 cells transfected with the miR-29a precursor or control virus. * P<0.05. (B) Notch1, Hes1, and Nfia protein expression levels were detected using western blot analysis in TE-1 cells transfected with the miR-29a precursor virus or control virus. (C) TargetScan prediction of the miR-29a binding site within Nfia mRNA. (D) Map of the pmirGLO luciferase reporter vector. The red rectangles indicate the restrictive endonucleases used for cloning. (E) Luciferase activity assay: TE-1 cells were transfected with the miR-29a precursor or scrambled virus for four days, transfected with the reporter vectors for 24 h, and harvested. Protein extracts were prepared and assayed for firefly and Renilla luciferase activity, and firefly luciferase activity was normalized to Renilla luciferase activity. Data are presented as the mean ± standard deviation from three independent experiments. * P<0.05. miR, microRNA; Hes1, hairy and enhancer of split 1; Nfia, nuclear factor 1 A; UTR, untranslated region.

Article Snippet: The membranes were blocked with 5% non-fat milk in PBS and Tween 20 (PBST; Beyotime Institute of Biotechnology) for 1 h at room temperature and incubated with polyclonal rabbit anti-goat Nfia (sc-30918; 1:1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), polyclonal rabbit anti-goat Notch1 (sc-6014; 1:1,000; Santa Cruz Biotechnology, Inc.), polyclonal rabbit anti-goat Hes1 (sc-13842; 1:1,000; Santa Cruz Biotechnology, Inc.) or polyclonal rabbit anti-goat β-actin (sc-1616; 1:1,000; Santa Cruz Biotechnology, Inc.), at 4°C overnight.

Techniques: Over Expression, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Transfection, Control, Virus, Western Blot, Binding Assay, Luciferase, Plasmid Preparation, Cloning, Activity Assay, Standard Deviation

Journal: Oncology Letters

Article Title: miR-29a activates Hes1 by targeting Nfia in esophageal carcinoma cell line TE-1

doi: 10.3892/ol.2014.2678

Figure Lengend Snippet: Knockdown of Nfia in TE-1 cells increases Hes1 expression levels and inhibits cell growth. (A) Reverse transcription-quantitative polymerase chain reaction was performed to determine Nfia and Hes1 mRNA expression levels in Nfia-knockdown TE-1 cells and control cells. * P<0.05. (B) Western blot analysis was performed to determine Hes1 protein expression levels in Nfia-knockdown TE-1 cells and control cells. (C) Knockdown of Nfia inhibits TE-1 cell proliferation, as determined by flow cytometry. Data are presented as the mean ± standard deviation from three independent experiments. Nfia, nuclear factor 1 A; Hes1, hairy and enhancer of split 1.

Article Snippet: The membranes were blocked with 5% non-fat milk in PBS and Tween 20 (PBST; Beyotime Institute of Biotechnology) for 1 h at room temperature and incubated with polyclonal rabbit anti-goat Nfia (sc-30918; 1:1,000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), polyclonal rabbit anti-goat Notch1 (sc-6014; 1:1,000; Santa Cruz Biotechnology, Inc.), polyclonal rabbit anti-goat Hes1 (sc-13842; 1:1,000; Santa Cruz Biotechnology, Inc.) or polyclonal rabbit anti-goat β-actin (sc-1616; 1:1,000; Santa Cruz Biotechnology, Inc.), at 4°C overnight.

Techniques: Knockdown, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Control, Western Blot, Flow Cytometry, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Asiatic Acid Attenuates Salmonella typhimurium -Induced Neuroinflammation and Neuronal Damage by Inhibiting the TLR2/Notch and NF-κB Pathway in Microglia

doi: 10.3390/ijms27020602

Figure Lengend Snippet: AA inhibits S.T -induced activation of the NF-κB and TLR2/Notch pathway in hippocampus. ( A ) Western blot to detect NF-κB protein levels in the hippocampus of mice with S.T stimulation and AA pretreatment. ( B ) Grayscale values of NF-κB pathway protein expression in mice hippocampi ( n = 3, * p < 0.05, compared with control; # p < 0.05, compared with S.T ). ( C ) Western blot to detect TLR2/Notch protein levels in the hippocampus of mice with S.T stimulation and AA pretreatment. ( D ) Grayscale values of TLR2/Notch pathway protein expression in mice hippocampi ( n = 3, * p < 0.05, compared with control; # p < 0.05, compared with S.T ). ( E ) qPCR detection of Notch1 , Notch2 and Hes1 mRNA expression in S.T -challenged or AA pretreatment hippocampus ( n = 3, * p < 0.05, compared with control; # p < 0.05, compared with S.T ). ( F ) Representative immunohistochemistry (IHC) images showing the Notch1 and Notch2 protein levels in mice hippocampi (scale = 100 μM).

Article Snippet: The following antibodies were used: anti-p65 (#8242, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-p65 (#3033, Cell Signaling Technology), anti-IκB (#76041, Cell Signaling Technology), anti-phospho-IκB (#2859, Cell Signaling Technology), anti-Notch1 NICD (#3608, Cell Signaling Technology), anti-Notch2 NICD (#5732, Cell Signaling Technology), anti-Iba1/AIF-1 (#17198, Cell Signaling Technology), Hes1 (#11988, Cell Signaling Technology), iNOS (18985, Proteintech, Wuhan, China) anti-β-actin (66009, Proteintech), Goat anti-Mouse IgG (H+L)-HRP (PR30012, Proteintech), Goat anti-Rabbli IgG (H+L)-HRP(PR30011, Proteintech), Goat anti-Rabbit lgG AF 488 ( M21012 , Abmart, Shanghai, China), Goat anti-Mouse lgG AF 594 ( M21013 , Abmart).

Techniques: Activation Assay, Western Blot, Expressing, Control, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Asiatic Acid Attenuates Salmonella typhimurium -Induced Neuroinflammation and Neuronal Damage by Inhibiting the TLR2/Notch and NF-κB Pathway in Microglia

doi: 10.3390/ijms27020602

Figure Lengend Snippet: Effect of AA on S.T -induced TLR2/Notch pathway proteins in BV-2 cells. ( A ) Activation of TLR2/Notch pathway in BV-2 cells after S.T infection and AA pretreatment detected by Western blot. ( B ) TLR2/Notch pathway protein grayscale images in BV-2 cells ( n = 3, * p < 0.05, compared with control; # p < 0.05, compared with S.T ). ( C ) Determination of Notch1 , Notch2 and Hes1 mRNA expression after S.T infection and pretreatment with AA by qPCR in microglia ( n = 3, * p < 0.05, compared with control; # p < 0.05, compared with S.T ). ( D ) Co-IP determination of protein interactions between Notch and p65 after S.T infection and AA pretreatment in microglia.

Article Snippet: The following antibodies were used: anti-p65 (#8242, Cell Signaling Technology, Danvers, MA, USA), anti-phospho-p65 (#3033, Cell Signaling Technology), anti-IκB (#76041, Cell Signaling Technology), anti-phospho-IκB (#2859, Cell Signaling Technology), anti-Notch1 NICD (#3608, Cell Signaling Technology), anti-Notch2 NICD (#5732, Cell Signaling Technology), anti-Iba1/AIF-1 (#17198, Cell Signaling Technology), Hes1 (#11988, Cell Signaling Technology), iNOS (18985, Proteintech, Wuhan, China) anti-β-actin (66009, Proteintech), Goat anti-Mouse IgG (H+L)-HRP (PR30012, Proteintech), Goat anti-Rabbli IgG (H+L)-HRP(PR30011, Proteintech), Goat anti-Rabbit lgG AF 488 ( M21012 , Abmart, Shanghai, China), Goat anti-Mouse lgG AF 594 ( M21013 , Abmart).

Techniques: Activation Assay, Infection, Western Blot, Control, Expressing, Co-Immunoprecipitation Assay