Additional file 2: of Identification of key genes in human airway epithelial cells in response to respiratory pathogens using microarray analysis

The GSE6802 mRNA expression profile dataset. 1241 DEGs between normal and infected airway epithelial cells were identified. (XLSX 101 kb

Additional file 3: of Identification of key genes in human airway epithelial cells in response to respiratory pathogens using microarray analysis

The GSE48466 mRNA expression profile dataset. 12950 DEGs between normal and infected airway epithelial cells were identified. (XLSX 964 kb

Beta-Elemene Blocks Epithelial-Mesenchymal Transition in Human Breast Cancer Cell Line MCF-7 through Smad3-Mediated Down-Regulation of Nuclear Transcription Factors

<div><p>Epithelial-mesenchymal transition (EMT) is the first step required for breast cancer to initiate metastasis. However, the potential of drugs to block and reverse the EMT process are not well explored. In the present study, we investigated the inhibitory effect of beta-elemene (ELE), an active component of a natural plant-derived anti-neoplastic agent in an established EMT model mediated by transforming growth factor-beta1 (TGF-β1). We found that ELE (40 µg/ml ) blocked the TGF-β1-induced phenotypic transition in the human breast cancer cell line MCF-7. ELE was able to inhibit TGF-β1-mediated upregulation of mRNA and protein expression of nuclear transcription factors (SNAI1, SNAI2, TWIST and SIP1), potentially through decreasing the expression and phosphorylation of Smad3, a central protein mediating the TGF-β1 signalling pathway. These findings suggest a potential therapeutic benefit of ELE in treating basal-like breast cancer.</p> </div

Forced expression of Rap1A accelerates osteoblast differentiation through activation of ERK1/2 and p38 MAPK.

<p><b>(a)</b> MC3T3-E1 cells stably expressing Rap1A were established as stated in the ‘‘Materials and Methods,” and Western blotting was used to test Rap1A expression with antibodies recognizing HA and Rap1A. <b>(b)</b> Proliferation of MC3T3-E1 cells with or without Rap1A overexpression tested by MTT assays. <b>(c-g)</b> Over-expression of Rap1A in MC3T3-E1 cells increased osteoblast-specific gene expression. <b>(h)</b> Western blot analyses with antibodies recognizing phospho-ERK1/2, total ERK1/2, phospho-p38, total p38 and Rap1A were performed in cells treated with either growth medium (GM) or osteoblast differentiation medium (DM) containing rhBMP-2 (100 ng/ml), ascorbic acid (50 μg/ml), and β-glycerophosphate (10 mM) for 3 days. β-actin was used as a loading control. <b>(i)</b> phospho-ERK1/2, total ERK1/2 and Rap1A were detected by Western blot in MC3T3-E1 cells with or without Rap1A overexpression after treatment with osteoblast differentiation medium (DM) for 3 days in the presence or absence of 10 μM U0126. β-actin was used as a loading control. <b>(j and k)</b> Inhibition of ALP activity by U0126 and SB203580 in cells with or without Rap1A overexpression after differentiation induction by osteogenic medium containing rhBMP-2 (100 ng/ml), ascorbic acid (50 μg/ml), and β-glycerophosphate (10 mM) for 3 days in the presence or absence of 10 μM U0126 <b>(j)</b> or 10 μM SB203580 <b>(k)</b>. Cells were fixed and stained for ALP. ALP activities were measured by densitometry at 520 nm (below). Data represent means ± SD of triplicate samples. *, P < 0.05; **, P < 0.01.</p

Expression of Rap1A during osteoblastic differentiation.

<p><b>(a)</b> Endogenous expression of Rap-1A in MC3T3-E1 and C2C12 cells by Western blotting. <b>(b)</b> The temporal change of Rap1A expression in MC3T3-E1 cells during osteoblastic differentiation by Western blotting. <b>(c)</b> Alizarin red staining for bone nodules in MC3T3-E1 cells. Original magnification was ×20 (left). Arizalin red-S staining activity was quantified by densitometry at 562 nm (right). Data represent means ± SD of triplicate samples. *P < 0.05, **P < 0.01 vs. the undifferentiated cells. β-actin was used as the internal control.</p

Association of Rap1A with osteoblast differentiation.

<p><b>(a)</b> Immunoblot analysis of Rap1A expression during osteogenic differentiation. The protein extracts were immunoblotted with anti-Rap1A antibody. β-Actin was used as the internal control. <b>(b-f)</b> Changes of osteoblastic markers gene expression in differentiated C2C12 cells. C2C12 cells were cultured with osteogenic medium containing ascorbic acid (50μg/ml), and β-glycerophosphate (10 mM) for the indicated times. Quantitative real-time PCR was performed for the mRNA expression of Rap1A, Runx2, Col1a1, Osteocalcin, and Osterix. <b>(g)</b> Alkaline phosphatase (ALP) staining and ALP activity measurement were determined during osteoblastic differentiation from C2C12 cells. <b>(h)</b> Osteoblastic mineralization of C2C12 cells was determined by Alizarin red staining and calcium content quantified in the cellular matrix by spectrophotometer (OD562nm). Data represent means ± SD of triplicate samples. *P<0.05, **P<0.01 vs. the undifferentiated cells.</p

TGF-β1 induces epithelial-to-mesenchymal changes in MCF-7 cells.

<p>(A) MCF-7 cells treated with 10 ng/ml TGF-β1 for 24 hrs had a spindle-like morphology and lost intercellular junctions. Magnification, 200×. (B) Western immunoblot analysis of expression of EMT-related proteins. Expression levels of E-cadherin and β-catenin (epithelial markers) in TGF-β1-treated MCF-7 cells were markedly decreased, whereas expression levels of N-cadherin and vimentin (mesenchymal markers) were dramatically increased. TGF-β1: transforming growth factor-β1.</p

ELE blocks TGF-β1-induced EMT in MCF-7 cells.

<p>(A) Representative pictures of MCF-7 cells treated with ELE showed that epithelial morphology of cells was maintained even in the presence of TGF-β1. Magnification, 200×. (B) Western immunoblot analysis showed that expression levels of E-cadherin and β-catenin (epithelial markers) and N-cadherin and vimentin (mesenchymal markers) did not differ in cells treated with TGF-β1 and ELE compared to cells with ELE only.</p

ELE facilitates TGF-β1-mediated downregulation of Smad3 expression but inhibits increased Smad3 phosphorylation.

<p>(A) RT-PCR analyses showed that TGF-β1 treatment led to downregulation of Smad3 mRNA expression, and ELE enhanced the reduction in Smad3 expression (*: P<0.01, **: P<0.001). (B) Western immunoblot analysis showed that the ratio of p-Smad3/total Smad3 was increased in a time-dependent manner with 10 ng/ml TGF-β1 (*:P<0.01), although expression levels of total Smad3 were slightly decreased. (C) TGF-β1-induced Smad3 phosphorylation was blocked by ELE and total Smad3 was further decreased in MCF-7 cells treated with TGF-β1 and ELE (*: P<0.01, **: P<0.001).</p

Silencing of Rap1A decreases ERK and p38 activation.

<p>Representative immunoblots for Rap1A expression and MAPK activation in control and Rap1A shRNA expressing C2C12 cells grown in basal or osteogenic medium containing ascorbic acid (50 μg/ml), and β-glycerophosphate (10 mM) for 6 and 10 d. Western blot analyses with antibodies recognizing Rap1A, phospho-ERK1/2, total ERK1/2, phospho-p38 and total p38 were performed. β-actin was used for internal control.</p