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