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: Experimental and Therapeutic Medicine

Article Title: MicroRNA-185 inhibits the proliferation and migration of HaCaT keratinocytes by targeting peroxisome proliferator-activated receptor β

doi: 10.3892/etm.2021.9797

Figure Lengend Snippet: Effect of PI3K inhibitor LY294002 on the proliferation and migration of HaCaT keratinocytes. HaCaT keratinocytes were treated with 50 µmol/l PI3K inhibitor LY294002 for 48 h. (A) The phosphorylation level of p-AKT at S473 and T308 and the p-AKT/AKT ratio was determined via western blot analysis. (B) Proliferation of HaCaT keratinocytes was assessed using Cell Counting Kit-8 assay. (C) Colony formation assay was employed to assess the number of colonies in HaCaT keratinocytes. (D) The number of migrated HaCaT keratinocytes was examined using Transwell assay. (E) The protein level of cyclin D1, CDK6, CDK4, MMP-2 and MMP-9 in HaCaT keratinocytes was determined via western blot analysis. * P<0.05 vs. control. p, phosphorylated.

Article Snippet: After blocking with 5% non-fat milk at room temperature for 2 h, the membranes were incubated with the following primary antibodies: Cyclin D1 (1:200; cat. no. ab16663), CDK6 (1:3,000; cat. no. ab151247), CDK4 (1:1,000; cat. no. ab95255), p-AKT-T308 (1:1,000; cat. no. ab8933), AKT (1:500; cat. no. ab8805) (all from Abcam), MMP-2 (1:2,000; cat. no. sc-10736), MMP-9 (1:2,000; cat. no. sc-10737), PPARβ (1:5,000; cat. no. sc-74440), integrin-linked kinase (ILK; 1:2,000; cat. no. sc-20019) (all from Santa Cruz Biotechnology, Inc.), phosphoinositide-dependent protein kinase 1 (PDK1, 1:2,000; cat. no. BA4499), p-AKT-S473 (1:1,000; cat. no. P00024-6), (all from Boster Biological Technology Co., Ltd.) or GAPDH (1:2,500; cat. no. ab9485; Abcam) at 4 ̊C overnight.

Techniques: Migration, Phospho-proteomics, Western Blot, Cell Counting, Colony Assay, Transwell Assay, Control

Journal: Experimental and Therapeutic Medicine

Article Title: MicroRNA-185 inhibits the proliferation and migration of HaCaT keratinocytes by targeting peroxisome proliferator-activated receptor β

doi: 10.3892/etm.2021.9797

Figure Lengend Snippet: Effect of miR-185 and PPARβ overexpression on the proliferation and migration of HaCaT keratinocytes. HaCaT keratinocytes were transfected with miR-185 and PPARβ overexpression plasmid or their NCs. (A) The protein level of PPARβ, ILK, PDK1, p-AKT-S473, p-AKT-T308 and AKT in HaCaT keratinocytes was determined via western blot analysis. (B) Proliferation of HaCaT keratinocytes was assessed using Cell Counting Kit-8 assay. (C) The number of colonies in HaCaT keratinocytes was determined using colony formation assay. (D) Transwell assay was used to assess the number of migrated HaCaT keratinocytes. (E) The protein level of cyclin D1, CDK6, CDK4, MMP-2 and MMP-9 in HaCaT keratinocytes was assessed via western blot analysis. * P<0.05. PPARβ, peroxisome proliferator-activated receptor β; ILK, integrin-linked kinase; PDK1, phosphoinositide-dependent protein kinase 1; miR, microRNA; NC, negative control; p, phosphorylated.

Article Snippet: After blocking with 5% non-fat milk at room temperature for 2 h, the membranes were incubated with the following primary antibodies: Cyclin D1 (1:200; cat. no. ab16663), CDK6 (1:3,000; cat. no. ab151247), CDK4 (1:1,000; cat. no. ab95255), p-AKT-T308 (1:1,000; cat. no. ab8933), AKT (1:500; cat. no. ab8805) (all from Abcam), MMP-2 (1:2,000; cat. no. sc-10736), MMP-9 (1:2,000; cat. no. sc-10737), PPARβ (1:5,000; cat. no. sc-74440), integrin-linked kinase (ILK; 1:2,000; cat. no. sc-20019) (all from Santa Cruz Biotechnology, Inc.), phosphoinositide-dependent protein kinase 1 (PDK1, 1:2,000; cat. no. BA4499), p-AKT-S473 (1:1,000; cat. no. P00024-6), (all from Boster Biological Technology Co., Ltd.) or GAPDH (1:2,500; cat. no. ab9485; Abcam) at 4 ̊C overnight.

Techniques: Over Expression, Migration, Transfection, Plasmid Preparation, Western Blot, Cell Counting, Colony Assay, Transwell Assay, Negative Control

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