Journal: Neural Regeneration Research

Article Title: 5-O-Methylvisammioside alleviates depression-like behaviors by inhibiting nuclear factor kappa B pathway activation via targeting SRC

doi: 10.4103/NRR.NRR-D-24-00714

Figure Lengend Snippet: Experimental overview. (A) Mice were pretreated with MeV and Flu for 11 days before administration of LPS for 3 days to establish the LPS-induced depression model. Flu, an antidepressant, served as the positive control ( n = 14). (B) In the second experiment, the SRC inhibitor PP2 was administered to the indicated groups by intraperitoneal injections for 14 days with LPS model establishment as described above. At the end of treatment, the mice underwent behavioral tests and were subsequently euthanized. Whole brain tissue, hippocampus, and serum were collected ( n = 20). ALB: Albumin; Flu: fluoxetine; FST: forced swimming test; IF: immunofluorescence; LPS: lipopolysaccharide; MeV: 5-O-methylvisammioside; OFT: open field test; SPT: sucrose preference test; TST: tail suspension test; WB: Western blot.

Article Snippet: Saline, MeV (4 mg/kg per day; RS00021020, Nature-standard, Shanghai, China), Flu (10 mg/kg per day; PHR1394-1g, Sigma Aldrich), and PP2 (2.5 mg/kg per day; HY-13805, MedChemExpress, Monmouth Junction, NJ, USA) were administered via intraperitoneal injection using a 1 mL syringe on days 1–14.

Techniques: Positive Control, Immunofluorescence, Suspension, Western Blot

Journal: Neural Regeneration Research

Article Title: 5-O-Methylvisammioside alleviates depression-like behaviors by inhibiting nuclear factor kappa B pathway activation via targeting SRC

doi: 10.4103/NRR.NRR-D-24-00714

Figure Lengend Snippet: Effects of SRC inhibition on NF-κB pathway protein expression and depression-like behaviors in mice with LPS-treated. (A–H) Representative blots of TLR4, p-NF-κB, NF-κB, IκBα, IL1β, p-SRC, and SRC and quantification of protein expressions ( n = 3–6). (I, J) Results from the open field test (OFT) ( n = 10). (K) Results from the tail suspension test ( n = 10). (L) Results from the forced swimming test ( n = 9). (M) Results from the sucrose preference test ( n = 8). (N) Serum ALB levels in mice ( n = 6). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 (one-way analysis of variance followed by Tukey’s post hoc test). ALB: Albumin; IBA1: ionized calcium-binding adapter molecule 1; IκBα: inhibitory subunit of NF-κB alpha; IL1β: interleukin 1 beta; LPS: lipopolysaccharide; NF-κB: nuclear factor kappa B; ns: not significant; p-NF-κB: phospho-NF-κB; PP2: SRC inhibitor; SRC: non-receptor tyrosine kinase Src; p-SRC: phospho-SRC; TLR4: Toll-like receptor 4.

Article Snippet: Saline, MeV (4 mg/kg per day; RS00021020, Nature-standard, Shanghai, China), Flu (10 mg/kg per day; PHR1394-1g, Sigma Aldrich), and PP2 (2.5 mg/kg per day; HY-13805, MedChemExpress, Monmouth Junction, NJ, USA) were administered via intraperitoneal injection using a 1 mL syringe on days 1–14.

Techniques: Inhibition, Expressing, Suspension, Binding Assay

Journal: Neural Regeneration Research

Article Title: 5-O-Methylvisammioside alleviates depression-like behaviors by inhibiting nuclear factor kappa B pathway activation via targeting SRC

doi: 10.4103/NRR.NRR-D-24-00714

Figure Lengend Snippet: Effects of MeV treatment on microglial activation in mice with LPS-induced depression. (A, B) Representative western blot images showing IBA1 expression and quantification of protein expression ( n = 6). (C, D) Representative fluorescence images of IBA1 (green) in the hippocampal CA1 and CA3 regions and quantification of relative intensity ( n = 3). Scale bars: 200 µm. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01 (one-way analysis of variance followed by Tukey’s post hoc test). CA: Cornu ammonis; IBA1: ionized calcium-binding adapter molecule 1; LPS: lipopolysaccharide; MeV: 5-O-methylvisammioside; PP2: SRC inhibitor.

Article Snippet: Saline, MeV (4 mg/kg per day; RS00021020, Nature-standard, Shanghai, China), Flu (10 mg/kg per day; PHR1394-1g, Sigma Aldrich), and PP2 (2.5 mg/kg per day; HY-13805, MedChemExpress, Monmouth Junction, NJ, USA) were administered via intraperitoneal injection using a 1 mL syringe on days 1–14.

Techniques: Activation Assay, Western Blot, Expressing, Fluorescence, Binding Assay

Journal: Scientific Reports

Article Title: Annexin A2 overexpression associates with colorectal cancer invasiveness and TGF-ß induced epithelial mesenchymal transition via Src/ANXA2/STAT3

doi: 10.1038/s41598-018-29703-0

Figure Lengend Snippet: The Src/ANXA2/STAT3 pathway regulates TGFB-induced EMT and cellular invasion. (A) DIC images representative of the cellular phenotype after TGF-ß treatment with or without pre-treatment with Src (PP2) and STAT3 (STA21) inhibitors. (B) Western blot for ANXA2 and Y23-pANXA2 after treatment of the cells with TGF-ß, PP2 or STA21, as indicated. ( C ) Western blot of ANXA2 expression after ANXA2 siRNA transfection. ( D ) Graph of Matrigel ® invasion assays: the number of invasive cells was normalized to the control in each group.

Article Snippet: STAT3 selective inhibitor (STA-21) and Src inhibitor (PP2) were bought from Santa Cruz and reconstituted in dimethyl sulfoxide (DMSO) at stock concentration of 10 mM, stored at −20 °C and used at a final concentration of 10 μM.

Techniques: Western Blot, Expressing, Transfection, Control

Journal: Blood

Article Title: Platelet-derived growth factor mediates survival of leukemic large granular lymphocytes via an autocrine regulatory pathway.

doi: 10.1182/blood-2009-06-223719

Figure Lengend Snippet: Figure 3. PDGF-BB mediates downstream target AKT/ ERK pathway activation via PI3K and SFK pathways in leukemic LGLs. (A) Western blot assay was carried out for SFK protein expression in PBMCs from 6 patients with T-LGL leukemia (LGL) and 6 normal controls (NL). The gels in the bottom panel indicate SFK expression of 60 KDa molecular weight. Kinase assay (top panel) was performed in IP samples to determine SFK activities (enolase). (B-C) Phospho-AKT and phospho-ERK (p44/42 MAPK) expression was determined in PBMC lysates from a representative patient with T-LGL leuke- mia (B) and a patient with NK-LGL leukemia (C) using Western blot assay. Western blot assay for GAPDH expression was performed to confirm equal loading of total protein in each lane. (D-G) Densitometry analysis was performed on Western blot results from 3 patients with T-LGL leukemia (D,F) and 3 patients with NK-LGL leukemia (E,G) to determine the average level of phospho-AKT expression (D-E) or phospho-ERK expres- sion (F-G). Data expressed as relative expression units of p-AKT/GAPDH or p-ERK/GAPDH ratios. (H) Western blot assay was performed for phospho-AKT protein expression in lysates of CD8 cells from a representative patient with T-LGL leukemia. Cells received different treatments as indicated. (Lane 1) Medium only. (Lane 2) Ten percent pooled sera from 3 patients with T-LGL leukemia. (Lanes 3,6) Ten percent pooled sera from either T-LGL leukemia patients or normal controls that received 2-hour preincubation with anti–PDGF-BB neutralizing antibody. (Lane 4) Ten percent pooled sera from patients with T-LGL leukemia that received 2-hour preincubation with IgG isotype control antibody. (Lane 5) Ten percent pooled sera from 3 normal controls. Western blot analysis for GAPDH was performed to confirm equal loading of total protein in each lane. (I) Densitometry was performed for Western blot results from 3 different experi- ments to determine the average level of phospho-AKT protein expression. *P .03, no treatment control (NTC) versus 10% T-LGL patient sera treatment; **P .05, treatment with 10% patient sera versus treatment with 10% patient sera preincubated with anti–PDGF-BB neu- tralizing antibody.

Article Snippet: Antibodies and inhibitors were obtained from the following sources and used at the dilutions recommended by the manufacturers: anti–PDGFR- (951) and anti–PDGFR- (958) polyclonal antibodies, anti–phospho-Tyr monoclonal antibody (PY99), goat anti–mouse immunoglobulin G (IgG) antibody (Santa Cruz Biotechnology Inc); anti–PDGF-BB neutralizing antibody and anti-PDGF-BB antibody for immunocytochemistry/immunofluorescence (ICC/IF; Abcam Inc); anti–phospho-AKT and total AKT polyclonal antibodies, anti-MEK1/2, anti–phospho-MEK1/2, anti-ERK1/2, and phospho-ERK1/2 (Cell Signaling Technology); anti– phospho-Src (Tyr419) and anti-Src antibodies (Upstate Cell Signaling Solutions); -actin monoclonal antibody (Sigma-Aldrich); mouse anti– glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (Chemicon International); PI3K inhibitor LY294002 (Cell Signaling Technology); Src family kinase (SFK) inhibitor PP2 and PDGF receptor tyrosine kinase inhibitor AG1296 (Calbiochem-Novabiochem Corp).

Techniques: Activation Assay, Western Blot, Expressing, Molecular Weight, Kinase Assay, Control

Journal: Blood

Article Title: Platelet-derived growth factor mediates survival of leukemic large granular lymphocytes via an autocrine regulatory pathway.

doi: 10.1182/blood-2009-06-223719

Figure Lengend Snippet: Figure 4. Inhibition of PDGF or LGL patient sera stimulation of NK leukemia cell line growth by blockade of PI3K/SFK or neutralizing antibody to PDGF-BB, respectively. (A-B) NKL cells were treated with rhPDGF-BB in presence of AG1296 at different concentrations (A), or in presence or absence of LY294002, PP2, or the combination of these 2 compounds (B); cell proliferation was measured by MTT assay. (C-D) NKL cells were treated with 10% pooled sera from patients with T-LGL leukemia (C) or from patients with NK-LGL leukemia (D) for 24 hours; cell proliferation was determined by MTT assay. Ten percent pooled sera sample from normal donors was used as controls. *P .01 for cell viability in samples receiving 10% normal sera treatment versus samples receiving 10% LGL patient sera treatment; **P .03 for cell viability in the samples receiving 10% patient sera treatment versus samples receiving treatment of 10% patient sera preincubated with anti–PDGF-BB neutralizing antibody. Rabbit IgG antibody was used as isotype control.

Article Snippet: Antibodies and inhibitors were obtained from the following sources and used at the dilutions recommended by the manufacturers: anti–PDGFR- (951) and anti–PDGFR- (958) polyclonal antibodies, anti–phospho-Tyr monoclonal antibody (PY99), goat anti–mouse immunoglobulin G (IgG) antibody (Santa Cruz Biotechnology Inc); anti–PDGF-BB neutralizing antibody and anti-PDGF-BB antibody for immunocytochemistry/immunofluorescence (ICC/IF; Abcam Inc); anti–phospho-AKT and total AKT polyclonal antibodies, anti-MEK1/2, anti–phospho-MEK1/2, anti-ERK1/2, and phospho-ERK1/2 (Cell Signaling Technology); anti– phospho-Src (Tyr419) and anti-Src antibodies (Upstate Cell Signaling Solutions); -actin monoclonal antibody (Sigma-Aldrich); mouse anti– glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (Chemicon International); PI3K inhibitor LY294002 (Cell Signaling Technology); Src family kinase (SFK) inhibitor PP2 and PDGF receptor tyrosine kinase inhibitor AG1296 (Calbiochem-Novabiochem Corp).

Techniques: Inhibition, MTT Assay, Control

Journal: Blood

Article Title: Platelet-derived growth factor mediates survival of leukemic large granular lymphocytes via an autocrine regulatory pathway.

doi: 10.1182/blood-2009-06-223719

Figure Lengend Snippet: Figure 5. Inhibition of PI3K and SFK pathways induces apoptosis in PBMCs from patients with LGL leukemia. (A-B) Freshly isolated PBMCs from a representative patient with NK-LGL leukemia (A) or a patient with T-LGL leukemia (B) were treated with LY129004 (PI3K inhibitor) or PP2 (SFK inhibitor) at doses of 10, 25, or 50M/L, respectively, and with the combination of both inhibitors at doses of 10 and 25M/L concentrations for 24 or 48 hours, respectively. DMSO served as vehicle control. (C) PBMCs from 5 patients with T-LGL leukemia (patients 1-5), from 4 patients with NK-LGL leukemia (patients 6-9), and from 4 normal controls (NLs 10-13) were treated with LY129004 or SFK PP2 at a concentration of 25M, or with the combination of LY and PP2 at 25M for 24 hours. Apoptosis was determined by flow cytometry assay with annexin-V–FITC/7-amino-actinomycin D staining.

Article Snippet: Antibodies and inhibitors were obtained from the following sources and used at the dilutions recommended by the manufacturers: anti–PDGFR- (951) and anti–PDGFR- (958) polyclonal antibodies, anti–phospho-Tyr monoclonal antibody (PY99), goat anti–mouse immunoglobulin G (IgG) antibody (Santa Cruz Biotechnology Inc); anti–PDGF-BB neutralizing antibody and anti-PDGF-BB antibody for immunocytochemistry/immunofluorescence (ICC/IF; Abcam Inc); anti–phospho-AKT and total AKT polyclonal antibodies, anti-MEK1/2, anti–phospho-MEK1/2, anti-ERK1/2, and phospho-ERK1/2 (Cell Signaling Technology); anti– phospho-Src (Tyr419) and anti-Src antibodies (Upstate Cell Signaling Solutions); -actin monoclonal antibody (Sigma-Aldrich); mouse anti– glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibody (Chemicon International); PI3K inhibitor LY294002 (Cell Signaling Technology); Src family kinase (SFK) inhibitor PP2 and PDGF receptor tyrosine kinase inhibitor AG1296 (Calbiochem-Novabiochem Corp).

Techniques: Inhibition, Isolation, Control, Concentration Assay, Cytometry, Staining