Journal: Journal of Neuroinflammation

Article Title: Vagus nerve stimulation modulates hippocampal inflammation caused by continuous stress in rats

doi: 10.1186/s12974-022-02396-z

Figure Lengend Snippet: aVNS upregulates the expression of α7 nAChR and ChAT in the hippocampus of CS animals. A RT-PCR analysis of α7 nAChR mRNA expression in the hippocampus. Upper images of agarose gel electrophoresis are the representatives of four independent experiments. Quantification of DNA band intensity relative to actin is plotted in lower panel. B Western blot analysis of α7 nAChR in the hippocampal tissues. C Immunofluorescence images of α7 nAChR signals in the granule cell layer (GCL) of the dentate gyrus (DG) and CA3 and CA1 pyramidal cell layers. The nuclei of neurons were visualized by staining with Hoechst 33258 (blue). D Western blot analysis of ChAT protein in the hippocampal tissue. In B and D , upper images are the representatives from four independent experiments. Quantification of band intensity relative to actin is plotted (lower panel). ** p < 0.01, *** p < 0.001 (One-way ANOVA)

Article Snippet: Immunoblotting was performed using primary antibodies including anti-TNF-α (Rabbit-polyclonal, 1:2000; Abcam, Cambridge, UK), anti-IL-1β (Rabbit-polyclonal, 1:2000; Abcam), anti-IL-6 (Mouse-monoclonal, 1:2000; Abcam), anti-α7 nicotinic acetylcholine receptor (Rabbit-polyclonal, 1:200, Alomone Labs, Jerusalem, Israel), anti-choline acetyltransferase (Rabbit-monoclonal, 1:2000; Abcam), anti-5HT1A receptor (Rabbit-polyclonal, 1:1000, Abcam), anti-Iba-1 (Mouse-monoclonal, 1:1000; Fujifilm, Minato, Japan), anti-cleaved Caspase-3 (Rabbit-polyclonal, 1:1000; Cell Signaling Technology, Beverly, MA, USA) and anti-β-actin (Mouse-monoclonal, 1:50,000; Sigma-Aldrich) antibodies.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Western Blot, Immunofluorescence, Staining

Journal: Journal of Neuroinflammation

Article Title: Vagus nerve stimulation modulates hippocampal inflammation caused by continuous stress in rats

doi: 10.1186/s12974-022-02396-z

Figure Lengend Snippet: Effects of α7 nAChR inhibitors on the hippocampal production of inflammatory cytokines. An α7 nAChR inhibitor MLA or saline vehicle solution (Veh) was administered 20 min prior to aVNS in CS animals, and proteins were extracted from hippocampal tissues for western blot analysis. Upper images in A – C are the representatives of 4–5 independent experiments for IL-1β, IL-6, and TNF-α proteins. Quantification of protein band intensity relative to actin is plotted in lower panels. ** p < 0.01, *** p < 0.001 (One-way ANOVA)

Article Snippet: Immunoblotting was performed using primary antibodies including anti-TNF-α (Rabbit-polyclonal, 1:2000; Abcam, Cambridge, UK), anti-IL-1β (Rabbit-polyclonal, 1:2000; Abcam), anti-IL-6 (Mouse-monoclonal, 1:2000; Abcam), anti-α7 nicotinic acetylcholine receptor (Rabbit-polyclonal, 1:200, Alomone Labs, Jerusalem, Israel), anti-choline acetyltransferase (Rabbit-monoclonal, 1:2000; Abcam), anti-5HT1A receptor (Rabbit-polyclonal, 1:1000, Abcam), anti-Iba-1 (Mouse-monoclonal, 1:1000; Fujifilm, Minato, Japan), anti-cleaved Caspase-3 (Rabbit-polyclonal, 1:1000; Cell Signaling Technology, Beverly, MA, USA) and anti-β-actin (Mouse-monoclonal, 1:50,000; Sigma-Aldrich) antibodies.

Techniques: Western Blot

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-knockdown in RF/6A cells by siIGFBP-rP1. (A) Reverse transcription-quantitative PCR analysis of IGFBP-rP1 transcript expression in RF/6A cells. GAPDH served as an internal reference for control. Both siIGFBP-rP1 duplex 1 and 2 significantly inhibited the expression IGFBP-rP1 compared with controls ( # P<0.05). Furthermore, the inhibitory effect of siIGFBP-rP1 duplex 2 was significantly increased compared with siIGFBP-rP1 duplex 1 (*P<0.05). (B) IGFBP-rP1 expression was measured by western blotting and normalized to that of β-actin. The inhibitory effect of siIGFBP-rP1 on the IGFBP-rP1 protein expression was consistent with that of the RNA expression. The membranes were stripped off and probed for the proteins. Data are presented as the mean ± standard deviation of three independent experiments with similar results and calculated as the integrated optical density of IGFBP-rP1 relative to the internal reference. # P<0.01 vs. the blank control group. *P<0.05 vs. the siIGFBP-rP1 duplex 1 group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA. Lane M, marker; lane 1, blank control; lane 2, transfection reagent; lane 3, scrambled control siRNA; lane 4, siRNA duplex 1; lane 5, siRNA duplex 2.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Knockdown, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot, RNA Expression, Standard Deviation, Binding Assay, Marker, Transfection

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell growth curves of siIGFBP-rP1-transfected cells and untransfected cells cultured in normoxic conditions for 6, 12, 24, 48 and 72 h. The OD values of transfected cells at 12, 24 and 48 h were significantly higher compared with untransfected cells (*P<0.01). Furthermore, the OD value of siIGFBP-rP1 duplex 2-transfected cells was significantly higher compared with siIGFBP-rP1 duplex 1-transfected cells at 24 h ( # P<0.01). No significant differences were observed among the groups at 6 and 72 h. OD values are presented as the mean ± standard deviation of 4 wells/group and experiments were performed in triplicate. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA; OD, optical density.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Standard Deviation, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell growth curves of siIGFBP-rP1-transfected or untransfected cells cultured under normoxic or hypoxic conditions for 6, 12, 24, 48 and 72 h, as detected by MTS colorimetric assays. The OD values of hypoxic groups (CoCl 2 and 1% O 2 ) decreased significantly at 12, 24, 48 and 72 h compared with the control group ( # P<0.01). There was no significant difference between the hypoxic groups (P>0.05). The OD value of the siRNA group were significantly increased at 12, 24 and 48 h compared with controls (**P<0.01). Furthermore, the OD values of the transfected cells cultured in hypoxic conditions (CoCl 2 + siRNA group and 1% O 2 + siRNA group) for 12, 24, 48 and 72 h were significantly lower compared with the siRNA group; additionally, the values were significantly higher compared with the hypoxic groups (*P<0.01). No significant differences were identified between the CoCl 2 + siRNA, 1% O 2 + siRNA and control groups (P>0.05). OD values are presented as the mean ± standard deviation of 4 wells/group and experiments were performed in triplicate. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA; CoCl 2 , cobalt chloride; OD, optical density.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Control, Standard Deviation, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: Cell motility of siIGFBP-rP1-transfected or untransfected cells cultured under normoxic or hypoxic conditions for 24 h is detected by wound and Transwell assays. (A) Representative images showing that RF/6A cells migrated across the wound boundary to the blank area (light microscopy; magnification, ×400) and (B) passed across the filter toward the lower surface (light microscopy; magnification, ×200). Hypoxia significantly promoted cell mobility compared with the controls ( # P<0.01). siIGFBP-rP1 transfection (siIGFBP-rP1 group) further enhanced cell migration, as compared with untransfected cells cultured in hypoxic conditions (hypoxia group; *P<0.01). The transfected cells cultured in hypoxic conditions (hypoxia + siRNA group) exhibited a significantly increased migration ability compared with the transfected cells cultured in normoxic conditions (siIGFBP-rP1 group; **P<0.01). Values are presented as the mean ± standard deviation of 4 samples/group and experiments were performed in triplicate and are quantified as the ratio relative to the control group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Transfection, Cell Culture, Light Microscopy, Migration, Standard Deviation, Control, Binding Assay

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-silencing stimulates hypoxia-induced tube formation of RF/6A cells. Representative images (inverted phase contrast microscopy; magnification, ×50) demonstrating that RF/6A cells formed capillary-like tube structures within the Matrigel layer in different mediums. RF/6A cells in hypoxic conditions or siIGFBP-rP1-transfected cells significantly formed completely enclosed capillary-like tubes compared with the controls ( # P<0.01 the hypoxia group vs. the control group; *P<0.01 the siIGFBP-rP1 group vs. the control group). Moreover, siIGFBP-rP1 transfection further promoted tube formation in RF/6A cells in the hypoxia + siIGFBP-rP1 group compared with the siIGFBP-rP1 group (**P<0.01). The values are presented as the mean ± standard deviation of 4 samples/group and experiments were performed in triplicate and are quantified as the ratio relative to the control group. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific small interfering RNA.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Microscopy, Transfection, Control, Standard Deviation, Binding Assay, Small Interfering RNA

Journal: Molecular Medicine Reports

Article Title: IGFBP-rP1-silencing promotes hypoxia-induced angiogenic potential of choroidal endothelial cells via the RAF/MEK/ERK signaling pathway

doi: 10.3892/mmr.2020.11578

Figure Lengend Snippet: IGFBP-rP1-silencing upregulates the hypoxia-induced RAF/MEK/ERK signaling pathway activation and VEGF expression. Representative images and quantified data demonstrated that hypoxic stress upregulated B-RAF, p-MEK, p-ERK and VEGF expression in RF/6A cells compared with controls ( # P<0.05). siIGFBP-rP1 transfection significantly promoted hypoxia-induced B-RAF, p-MEK, p-ERK and VEGF expression in RF/6A compared with the hypoxia group ( # P<0.05). IGFBP-rP1 restoration significantly downregulated the expression of B-RAF, p-MEK, p-ERK and VEGF in siIGFBP-rP1-transfected cells, under both normoxic and hypoxic conditions, compared with the siIGFBP-rP1 and hypoxia + siIGFBP-rP1 groups, respectively (*P<0.01). The membranes were stripped off and probed for the proteins. Values are presented as the mean ± SD of 3 independent experiments with similar results and are presented as the integrated optical density of studied proteins relative to GAPDH. IGFBP-rP1, insulin-like growth factor binding protein-related protein 1; siIGFBP-rP1, IGFBP-rP1 specific siRNA. Lane 1, control; lane 2, siIGFBP-rP1; lane 3, siIGFBP-rP1 + IGFBP-rP1; lane 4, hypoxia; lane 5, hypoxia + siIGFBP-rP1; lane 6, hypoxia + siIGFBP-rP1 + IGFBP-rP1.

Article Snippet: Recombinant human IGFBP-rP1 and goat anti-human IGFBP-rP1 polyclonal antibody (cat. no. AF1334) were obtained from R&D Systems, Inc. Rabbit anti-human GAPDH polyclonal (cat. no. 10494-1-AP) and mouse anti-human β-actin monoclonal (cat. no. 66009-1-Ig) antibodies were obtained from ProteinTech Group, Inc. Rabbit anti-human VEGF polyclonal (cat. no. ab150766) antibodies were purchased from Abcam.

Techniques: Activation Assay, Expressing, Transfection, Binding Assay, Control

Journal: Frontiers in Genetics

Article Title: A Novel Long Noncoding RNA, lncR-125b, Promotes the Differentiation of Goat Skeletal Muscle Satellite Cells by Sponging miR-125b

doi: 10.3389/fgene.2019.01171

Figure Lengend Snippet: lncR-125b promotes myogenic differentiation in vitro . (A) Real-time PCR analysis of lncR-125b expression in SMSCs transfected with pEGFP-control or pEGFP-lncR-125b. (B) Real-time PCR analysis of MyoG and MyoD expression in SMSCs transfected with pEGFP-control or pEGFP-lncR-125b. (C) Real-time PCR analysis of lncR-125b expression in SMSCs transfected with siRNA-control or siRNA-lncR-125b. (D) Real-time PCR analysis of MyoG and MyoD expression in SMSCs transfected with siRNA-control or siRNA-lncR-125b. (E) MyHC immunostaining of SMSCs transfected with pEGFP-control vector or pEGFP-lncR-125b then cultured in differentiation medium (DM) for 7 days. The nuclei were visualized with DAPI (blue). Scale bar: 100 μm. (F) MyHC immunostaining of SMSCs transfected with siRNA-control or siRNA-lncR-125b then cultured in DM for 7 days. The nuclei were visualized with DAPI (blue). Scale bar: 100 μm. (G) Analysis of MyHC-staining cells after overexpression of lncR-125b. The percentage of MyHC positive cell was calculated as the ratio of the number of nuclei surrounded by MyHC signal to the total nuclei. (H) Analysis of MyHC-staining cells after knockdown of lncR-125b. The percentage of MyHC positive cell was calculated as the ratio of the number of nuclei surrounded by MyHC signal to the total nuclei. All data are shown as mean ± SEM of three biological replicates, * P < 0.05.

Article Snippet: Finally, the purified SMSCs were qualified by direct immunostaining with Pax7 (Paired box 7, rabbit anti-Pax7, 1:100 dilution; Boster, Wuhan, Hubei, China), a critical marker for SMSCs.

Techniques: In Vitro, Real-time Polymerase Chain Reaction, Expressing, Transfection, Control, Immunostaining, Plasmid Preparation, Cell Culture, Staining, Over Expression, Knockdown

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Acupuncture Alters Expression of Insulin Signaling Related Molecules and Improves Insulin Resistance in OLETF Rats

doi: 10.1155/2016/9651592

Figure Lengend Snippet: Effects of acupuncture on protein expressions of PI3K-p85, phospho-PKC ζ / λ , and GLUT4 in skeletal muscle of OLETF rats. Protein expression was determined by Western blot. GAPDH was used as an internal control. Data are shown as mean ± SD ( n = 8 each group). ∗ P < 0.05 and ∗∗ P < 0.01 versus SD group; △△ P < 0.01 versus OLETF group.

Article Snippet: Nonspecific binding sites were blocked with 5% milk powder diluted in TBS with 0.05% Tween 20 (TBST) for 60 min. Proteins were detected using the following antibodies: rabbit polyclonal antibody for PI3K-p85 (diluted 1 : 3000; Bios; bs-0128R), rabbit polyclonal antibody for phospho-PKC ζ / λ (diluted 1 : 3000; CST; #9378), rabbit polyclonal antibody for GLUT4 (diluted 1 : 3000; Boster; BA1626), and mouse monoclonal antibody for GAPDH (diluted 1 : 3000; Boster; BM1623).

Techniques: Expressing, Western Blot, Control