Bovine milk changes cell morphology.

<p>Normal human skin fibroblasts were cultured in regular medium without FCS or in the presence of 10% FCS, 1%, 5%, 10% and 20% bovine milk for 24 hours. Representative photographs are shown. Bar = 200μM</p

Bovine milk activates signaling molecules.

<p>Time course of PKB/Akt, p44/42 and p38 phosphorylation in human fibroblasts. After serum starvation for 24 hours cells were treated with either 10% FCS or 10% bovine milk for 10, 30, 60 minutes or 4 hours. Cells held under FCS-free conditions served as control (ctr). Proteins were obtained as described in “Materials and Methods”, and Western blotting was performed with phosphospecific antibodies. Equal loading was monitored by using antibodies directed against total PKB/Akt. The blot shows representative results. (n = 3).</p

TGFβ1 regulates collagen synthesis.

<p>Human skin fibroblasts were treated <b>(a)</b> with 10% bovine milk or with 0.01, 0.05, 0.1, 0.5, 1.0 ng/ml TGFβ1 or <b>(b)</b> with 10% milk containing 5, 10, 20 μg/ml TGFβ1,2,3 neutralizing antibody MAB1835. Cells held under FCS-free conditions served as control. After 48 h the amount of P1NP in the supernatants was measured. Each bar represents the mean of 3 independent experiments. Standard deviations are indicated. Data of (a) were compared to cells held under FCS-free conditions, and (b) treated with 10% milk and 20 μg/ml mIgG. *p<0.05</p

Bovine milk stimulates collagen synthesis and promoter transactivation.

<p>Normal human skin fibroblasts were cultured in medium supplemented with increasing concentrations of <b>(a)</b> bovine milk or <b>(b)</b> FCS for control. After 48 hours the amount of P1NP in the supernatants was measured. <b>(c)</b> Analysis of two collagen 1A1 promoter-based luciferase constructs (-2300/+18, -205/+18) in human fibroblasts. After transfection cells were treated with 10% bovine milk or TGFβ1 as positive control for 48 hours. Cells held under FCS-free conditions served as control. Transfection efficiacy was controlled by co-transfection with a humanized Renilla luciferase vector (phRL). Each bar represents the mean of 3 independent experiments. Standard deviations are indicated. Data were compared to untreated controls. *p<0.05</p

Impact of IGF-1, insulin and testosterone on collagen synthesis.

<p>Human skin fibroblasts were treated <b>(a)</b> with 10% bovine milk or with 10, 50, 100 ng/ml IGF-1, 6, 30, 60ng/ml insulin or with 75, 200, 400pg/ml testosterone or <b>(b)</b> with 10% bovine milk containing 5 and 10 μg/ml IGF-1 neutralizing antibody #5119–100 or <b>(c)</b> with 10% bovine milk containing 0.5 and 1 μg/ml MA-20, a functional blocking antibody against the insulin receptor. Cells held under FCS-free conditions served as control. After 48 hours the amount of P1NP in the supernatants was measured. Each bar represents the mean of 3 independent experiments. Standard deviations are indicated. Data of (a) were compared with cells held under FCS-free conditions, and (b,c) treated with 10% milk and mIgG. *p<0.05</p

Bovine milk regulates collagen synthesis via STAT6.

<p><b>(a)</b> Human skin fibroblasts were treated with 1, 5, 10 and 20% bovine milk with or without 200 μM leflunomide, an inhibitor of p-STAT6. After 0,5, 2 and 6h cell lysates were examined for p-STAT6 induction by Western blot. <b>(b)</b> Cells were treated with 1, 5, 10 and 20% bovine milk with or without 200μM leflunomide. After 24 hours the amount of P1NP in the supernatants was measured. Each bar represents the mean of 3 independent experiments. Standard deviations are indicated. Data were compared to cells held under FCS-free conditions. *p<0.05. <b>(c)</b> Cells were treated with 20% bovine milk in the presence of specific kinase inhibitors (20 μM LY294002, 500 nM AG1478, 100 μM PD98059, 20 μM SB203580). After 6h cell lysates were examined for p-STAT6 induction. <b>(d)</b> Cells were treated with either 20% bovine milk or with increasing concentrations of IGF-1 or insulin. After 6h cell lysates were examined for p-STAT6 induction. Equal loading was monitored by using antibodies directed against actin. The blots show representative results. (n = 3).</p

Additional file 1: Figure S1. of The histone deacetylase inhibitor trichostatin a decreases lymphangiogenesis by inducing apoptosis and cell cycle arrest via p21-dependent pathways

Effects of TSA on important cell cycle regulators. Densitometric analysis of Western blots. TSA induced the expression of p21, p27 and p53 in a concentration-dependent manner. LECs that were left untreated (solvent only) or were treated with TSA in a concentration-dependent manner for 24 h as indicated. The western blot bands were quantified by densitometry; absorbencies of p21, p27 and p53 bands were corrected for loading differences based on the corresponding tubulin bands. Experiments were performed at least three times, and the results are shown as mean ± SD. *p < 0.05 vs ctrl., ns = not significant. (JPG 28 kb