Journal: International journal of molecular sciences

Article Title: Improved Wound Healing and Skin Regeneration Ability of 3,2'-Dihydroxyflavone-Treated Mesenchymal Stem Cell-Derived Extracellular Vesicles.

doi: 10.3390/ijms24086964

Figure Lengend Snippet: Figure 2. Characterization of 3,2′-DHF-treated WJ-MSCs and EVs. (A) The proliferation rate of WJ- MSC treated with various concentrations of 3,2′-DHF. WJ-MSCs have the best proliferation rate with 1–2 µM 3,2′-DHF. Statistical analysis was determined using one-way ANOVA with Tukey’s multiple comparisons test: ** p < 0.05, **** p < 0.0001. (B) Protein expression of MEK/ERK signaling proteins in WJ-MSCs with and without 3,2′-DHF treatment. The bar graph shows the protein expression of AKT and MEK in 3,2′-DHF-treated and untreated WJ-MSCs. (C) Flow cytometry analysis to confirm the characteristics of WJ-MSCs with or without 3,2′-DHF. The expression of the positive markers CD73, CD90, and CD105, and the negative markers CD34 and CD45, was assessed. (D) The relative EV production amounts of Cont-EV and Fla-EV were measured using nanoparticle tracking analysis (NTA). Statistical analysis was determined using two-way ANOVA: **** p < 0.0001. (E) Transmission electron microscopy (TEM) images of Cont-EV and Fla-EV. Scale bar: 100 nm. The size of the EVs was determined with NTA. (F) Characterization of EV-positive markers CD9 and CD63, and negative markers calnexin and GM130, in Cont-EV and Fla-EV using Western blotting. (G) Representative plot of EV-surface markers CD63 and CD81 in Cont-EV and Fla-EV via flow cytometry analysis.

Article Snippet: The following antibodies were diluted in 1× blocking buffer (TLP-115.1G, Translab): CD9 (ab263023, Abcam, Cambridge, UK), CD63 (ab109201, Abcam), calnexin (2679, Cell Signaling Technology, Danvers, MA, USA), GM130 (12480, Santa Cruz Biotechnology), p-AKT (sc-293125, Santa Cruz Biotechnology), AKT (CSB-PA000855, Cusabio, Houston, TX, USA), p-ERK (CSB-PA000749, Cusabio), ERK (B7074, Tebu-bio, Le Perray, Ile-de-France, France), β-actin (sc-47778, Santa Cruz Biotechnology), HRP-linked anti-rabbit IgG (7074, Santa Cruz Biotechnology), and HRP-linked anti-mouse IgG (7076, Santa Cruz Biotechnology).

Techniques: Expressing, Flow Cytometry, Transmission Assay, Electron Microscopy, Western Blot, Cytometry

Journal: PLoS ONE

Article Title: The Anti-Inflammatory Effects and Mechanisms of Eupafolin in Lipopolysaccharide-Induced Inflammatory Responses in RAW264.7 Macrophages

doi: 10.1371/journal.pone.0158662

Figure Lengend Snippet: (A) Chemical structure of eupafolin. RAW264.7 macrophages were pretreated with 1, 1.5, or 2 μg/ml of LPS for 24 h. The control group included cells grown in medium without LPS. (B) COX-2 and iNOS protein expression was determined by Western blot analysis. GAPDH or β-actin was processed in parallel as an internal control for protein loading. (C) The RAW264.7 macrophages were pretreated with 0, 20, 40, or 60 μM eupafolin for 1 h and then with 1 μg/ml of LPS for another 24 h. The COX-2 expression was analyzed by Western blotting (n = 4). (D) Immunofluorescence staining was performed to show the COX-2 expression. (n = 4) (E) The RAW264.7 macrophages were pretreated with 0, 20, 40, or 60 μM eupafolin for 1 h and then with 1 μg/ml of LPS for another 24 h. The iNOS expression was measured using Western blot analysis (n = 4). (F) The Griess assay was performed to evaluate the NO production. In B-C and E-F, the data are shown as the means ± SEM (n = 4). * P <0.05 vs. the untreated group; † P <0.05 vs. the LPS-treated group. The scale bars in D = 25 μm.

Article Snippet: Polyclonal rabbit IgGs against human GAPDH, β-actin, phospho/total-p38, phospho/total-ERK1/2, phospho/total-JNK, COX-2, and phospho/total -AKT and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG or anti-rabbit IgG antibodies were purchased from GeneTex (Irvine, CA, USA).

Techniques: Control, Expressing, Western Blot, Immunofluorescence, Staining, Griess Assay

Journal: PLoS ONE

Article Title: The Anti-Inflammatory Effects and Mechanisms of Eupafolin in Lipopolysaccharide-Induced Inflammatory Responses in RAW264.7 Macrophages

doi: 10.1371/journal.pone.0158662

Figure Lengend Snippet: The RAW264.7 macrophages were treated for 1 h with 30 μM of the MAPK inhibitors or 10 μM of the PI3K/AKT inhibitor and were then incubated with 1 μg/ml of LPS for 24 h. (A) COX-2 and (B) iNOS protein expression was determined by Western blot analysis. β-actin was processed in parallel as an internal control for protein loading. (C) NO was measured with the Griess assay, and (D) PGE 2 was measured with an ELISA assay. The data are shown as the means ± SEM (n = 5–8). * P <0.05 vs. the untreated control, † P <0.05 vs. the LPS-treated cells. ‡ P <0.05 vs. the LPS+EUP treated cells.

Article Snippet: Polyclonal rabbit IgGs against human GAPDH, β-actin, phospho/total-p38, phospho/total-ERK1/2, phospho/total-JNK, COX-2, and phospho/total -AKT and horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG or anti-rabbit IgG antibodies were purchased from GeneTex (Irvine, CA, USA).

Techniques: Incubation, Expressing, Western Blot, Control, Griess Assay, Enzyme-linked Immunosorbent Assay

Journal: Oncology Letters

Article Title: Downregulation of BIRC5 inhibits the migration and invasion of esophageal cancer cells by interacting with the PI3K/Akt signaling pathway

doi: 10.3892/ol.2018.8986

Figure Lengend Snippet: Relative expression levels of BIRC5 mRNA and protein in different cell lines with or without SC79 treatment. (A) The effect of SC79 treatment on the expression levels of p-AKT in different cell lines. (B) Relative mRNA expression levels of BIRC5 in different cell lines. (C) Representative western blot analysis results of BIRC5. (D) Relative protein expression levels of BIRC5 in different cell lines. (E) The effect of SC79 treatment on the expression levels of VEGF in different cell lines. (F) The effect of SC79 treatment on the expression levels of BAI1 in different cell lines. (G) The effect of BAI1 treatment on the expression levels of MBD2 in different cell lines. *P<0.05 vs. untreated cells. BIRC5, baculoviral IAP repeat containing 5; VEGF, vascular endothelial growth factor; MBD2, methyl-CpG binding domain protein 2; BIRC5, baculoviral IAP repeat containing 5; VEGF, vascular endothelial growth factor; BAI1, brain-specific angiogenesis inhibitor 1; MBD2, methyl-CpG binding domain protein 2; p-Akt, phospho-Akt.

Article Snippet: The membranes were then incubated with corresponding primary antibodies including rabbit anti-BIRC5 polyclonal antibody (dilution, 1:1,000; catalog no. MBS151070; MyBioSource, Inc., San Diego, CA, USA), rabbit anti-VEGF polyclonal antibody (dilution, 1:1,000; catalog no. bs-0279R; Biomathematics and Statistics Scotland, Edinburgh, Scotland), rabbit anti-BAI1 polyclonal antibody (dilution, 1:1,000; catalog no. MBS8242484; MyBioSource, Inc.), rabbit anti-MBD2 polyclonal antibody (dilution, 1:1,000; catalog no. MBS126361; MyBioSource, Inc.), rabbit anti-phospho (p)-Akt polyclonal antibody (dilution, 1:1,000; catalog no. MBS330024; MyBioSource, Inc.) and rabbit anti-GAPDH polyclonal antibody (dilution, 1:1,000; catalog no. MBS9216842; MyBioSource, Inc.) overnight at 4°C.

Techniques: Expressing, Western Blot, Binding Assay