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: The Journal of Endocrinology

Article Title: PI3 kinase directly phosphorylates Akt1/2 at Ser473/474 in the insulin signal transduction pathway

doi: 10.1530/JOE-13-0172

Figure Lengend Snippet: Insulin stimulates GLUT4 translocation toward the plasma membrane in an Akt1/2-dependent manner. (A) The Akt1 or Akt2 concentration/intensity standard curves were made using a human recombinant Akt1 or Akt2 respectively. 3T3-L1-GLUT4myc fibroblasts on day 14 after differentiation induction were lysed followed by western blotting using antibodies against Akt1 and Akt2, and the amount of Akt1 and Akt2 was calculated from each standard curve ( n =4 independent experiments). (B) 3T3-L1-GLUT4myc adipocytes were treated with insulin (100 nM) in the presence and absence of MK2206 (+MK) (5 μM). Then, cells were lysed and separated into the cytosolic (C) and plasma membrane fractions (M), followed by western blotting using an antibody against c-myc. In the graph, each column represents the mean (± s.e.m .) ratio of signal intensity for c-myc in the plasma membrane fraction to that in the total cell ( n =4 independent experiments). P values, ANOVA followed by a Bonferonni correction. NS, not significant. (C) In the left panel, 3T3-L1-GLUT4myc adipocytes were transfected with the NC siRNA or the Akt1/2 siRNA, and 48 h after transfection western blotting was carried out using antibodies against Akt1/2 or β-actin. Signal intensities for Akt1/2 were normalized to those for β-actin. In the graph, each column represents the mean (± s.e.m .) normalized expression of Akt1/2 ( n =4 independent experiments). P value, unpaired t -test. In the right panel, cells transfected with the NC siRNA (NC) or the Akt1/2 siRNA (Akt1/2 KD) were left untreated or treated with insulin (100 nM) for 20 min. Then, cells were lysed and separated into the cytosolic (C) and plasma membrane fractions (M), followed by western blotting using an antibody against c-myc. In the graph, each column represents the mean (± s.e.m .) ratio of signal intensity for c-myc in the plasma membrane fraction to that in the total cell ( n =4 independent experiments). P values, ANOVA followed by a Bonferonni correction. NS, not significant.

Article Snippet: Human recombinant Akt1 or human recombinant Akt2 (Active Motif, Carlsbad, CA, USA) was reacted without or with His-tagged human recombinant PI3K (p110β/p85α) (Sigma) or His-tagged human recombinant PDK1 (SignalChem, Richmond, BC, Canada), which was purified by affinity chromatography, in the presence and absence of wortmannin or BX912 in a medium containing 25 mM 3-morpholinopropanesulfonic acid (pH 7.2), 25 mM MgCl 2 , 12.5 mM glycerol 2-phosphate, 5 mM ethylene glycol-bis(2-aminoethyl ether)- N , N , N ′, N ′-tetraacetic acid, 2 mM EDTA, 0.25 mM dithiothreitol, and 250 μM ATP at 30 °C for 20 min. Phosphorylated Akt was detected by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2 as described earlier.

Techniques: Translocation Assay, Clinical Proteomics, Membrane, Concentration Assay, Recombinant, Western Blot, Transfection, Expressing

Journal: The Journal of Endocrinology

Article Title: PI3 kinase directly phosphorylates Akt1/2 at Ser473/474 in the insulin signal transduction pathway

doi: 10.1530/JOE-13-0172

Figure Lengend Snippet: Insulin stimulates phosphorylation of Akt1/2 in a PI3K-dependent manner in 3T3-L1-GLUT4myc adipocytes. Cells were left untreated or treated with insulin (Ins) (100 nM) for 10 min in the presence and absence of wortmannin (+WM) (20 nM) (A) or BX912 (+BX) (100 nM) (B). Western blotting was carried out using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1/2 (pAkt1/2) were normalized to those for Akt1/2. In the graphs, each column represents the mean (± s.e.m .) normalized intensity for pAkt1/2 at each site ( n =4 independent experiments). P values, ANOVA followed by a Bonferonni correction. NS, not significant.

Article Snippet: Human recombinant Akt1 or human recombinant Akt2 (Active Motif, Carlsbad, CA, USA) was reacted without or with His-tagged human recombinant PI3K (p110β/p85α) (Sigma) or His-tagged human recombinant PDK1 (SignalChem, Richmond, BC, Canada), which was purified by affinity chromatography, in the presence and absence of wortmannin or BX912 in a medium containing 25 mM 3-morpholinopropanesulfonic acid (pH 7.2), 25 mM MgCl 2 , 12.5 mM glycerol 2-phosphate, 5 mM ethylene glycol-bis(2-aminoethyl ether)- N , N , N ′, N ′-tetraacetic acid, 2 mM EDTA, 0.25 mM dithiothreitol, and 250 μM ATP at 30 °C for 20 min. Phosphorylated Akt was detected by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2 as described earlier.

Techniques: Phospho-proteomics, Western Blot

Journal: The Journal of Endocrinology

Article Title: PI3 kinase directly phosphorylates Akt1/2 at Ser473/474 in the insulin signal transduction pathway

doi: 10.1530/JOE-13-0172

Figure Lengend Snippet: Insulin-induced Akt1/2 phosphorylation at Thr308 and Ser473 was prevented by knocking-down PI3K, but not PDK1, in 3T3-L1-GLUT4myc adipocytes. (A) In the left panel, cells were transfected with the NC siRNA or the PI3K siRNA, and 48 h after transfection western blotting was carried out using antibodies against PI3K or β-actin. Signal intensities for PI3K were normalized to those for β-actin. In the graph, each column represents the mean (± s.e.m .) normalized expression of PI3K ( n =4 independent experiments). P value, unpaired t -test. In the right panel, cells transfected with the NC siRNA (NC) or the PI3K siRNA (PI3K KD) were left untreated or treated with insulin (Ins) (100 nM) for 10 min, followed by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1/2 (pAkt1/2) were normalized to those for Akt1/2. In the graph, each column represents the mean (± s.e.m .) normalized intensity for pAkt1/2 at each site ( n =4 independent experiments). P values, ANOVA followed by a Bonferonni correction. (B) In the left panel, cells were transfected with the NC siRNA or the PDK1 siRNA, and 48 h after transfection western blotting was carried out using antibodies against PDK1 or β-actin. Signal intensities for PDK1 were normalized to those for β-actin. In the graph, each column represents the mean (± s.e.m .) normalized expression of PDK1 ( n =4 independent experiments). P value, unpaired t -test. In the right panel, cells transfected with the NC siRNA (NC) or the PDK1 siRNA (PDK1 KD) were left untreated or treated with insulin (Ins) (100 nM) for 10 min, followed by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1/2 (pAkt1/2) were normalized to those for Akt1/2. In the graph, each column represents the mean (± s.e.m .) normalized intensity for pAkt1/2 at each site ( n =4 independent experiments). P values, ANOVA followed by a Bonferonni correction. NS, not significant.

Article Snippet: Human recombinant Akt1 or human recombinant Akt2 (Active Motif, Carlsbad, CA, USA) was reacted without or with His-tagged human recombinant PI3K (p110β/p85α) (Sigma) or His-tagged human recombinant PDK1 (SignalChem, Richmond, BC, Canada), which was purified by affinity chromatography, in the presence and absence of wortmannin or BX912 in a medium containing 25 mM 3-morpholinopropanesulfonic acid (pH 7.2), 25 mM MgCl 2 , 12.5 mM glycerol 2-phosphate, 5 mM ethylene glycol-bis(2-aminoethyl ether)- N , N , N ′, N ′-tetraacetic acid, 2 mM EDTA, 0.25 mM dithiothreitol, and 250 μM ATP at 30 °C for 20 min. Phosphorylated Akt was detected by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2 as described earlier.

Techniques: Phospho-proteomics, Transfection, Western Blot, Expressing

Journal: The Journal of Endocrinology

Article Title: PI3 kinase directly phosphorylates Akt1/2 at Ser473/474 in the insulin signal transduction pathway

doi: 10.1530/JOE-13-0172

Figure Lengend Snippet: PI3K-dependent and PDK1-independent Akt1 phosphorylation and PI3K-/PDK1-dependent Akt2 phosphorylation under cell-free conditions. Akt1 or Akt2 was reacted with (+) and without (−) PI3K (1 μg/ml) (A and C) or PDK1 (1 μg/ml) (B and D) in the presence and absence of wortmannin (WM) (20 nM) or BX912 (BX) (100 nM), and western blotting was carried out using antibodies against pT308(9), pS473(4), and Akt1/2. Signal intensities for phosphorylated Akt1 (pAkt1) or Akt2 (pAkt2) were normalized to those for Akt1 or Akt2. In the graphs, each value represents the mean (± s.e.m .) intensity for pAkt1 or pAkt2 at each site ( n =4). P values, Dunnett's test. NS, not significant.

Article Snippet: Human recombinant Akt1 or human recombinant Akt2 (Active Motif, Carlsbad, CA, USA) was reacted without or with His-tagged human recombinant PI3K (p110β/p85α) (Sigma) or His-tagged human recombinant PDK1 (SignalChem, Richmond, BC, Canada), which was purified by affinity chromatography, in the presence and absence of wortmannin or BX912 in a medium containing 25 mM 3-morpholinopropanesulfonic acid (pH 7.2), 25 mM MgCl 2 , 12.5 mM glycerol 2-phosphate, 5 mM ethylene glycol-bis(2-aminoethyl ether)- N , N , N ′, N ′-tetraacetic acid, 2 mM EDTA, 0.25 mM dithiothreitol, and 250 μM ATP at 30 °C for 20 min. Phosphorylated Akt was detected by western blotting using antibodies against pT308(9), pS473(4), and Akt1/2 as described earlier.

Techniques: Phospho-proteomics, Western Blot