Additional file 1 of Effect of neuromuscular electrical stimulation in critically ill adults with mechanical ventilation: a systematic review and network meta-analysis

Additional file 1:. Supplementary Tables and Figure

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

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 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

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

MCF-7 cells acquire migration and invasion abilities in response to TGF-β1 treatment.

<p>(A) Representative pictures of the scratch wound assay. TGF-β1-treated MCF-7 cells migrated faster than controls. The black lines indicated size of the remaining wound. Data from three independent experiments were analyzed and presented as mean ± SD. (P<0.001). (B) Representative pictures in a transwell migration assay (top).The penetrated cells numbers of MCF-7 treated with TGF-β1 for 12 hrs were significantly increased as compared to controls without TGF-β1 (P<0.001). Representative pictures in a transwell invasion assay (bottom). TGF-β1 treatment led to more MCF-7 cells penetrating matrix than controls. (P = 0.002). The results in both transwell migration and invasion assays were obtained from three independent experiments.</p

TGF-β1-mediated upregulation of nuclear transcriptional factors expression was blocked by ELE.

<p>(A) RT-PCR analyses showed that mRNA expression levels of transcriptional factors (SNAI1, SNAI2, TWIST and SIP1) were significantly increased in TGF-β1 treated MCF-7 cells (*: P<0.01, **: P<0.001). (B) Western immunoblot analysis showed that protein expression levels of SNAI1, SNAI2, TWIST and SIP1 were repressed by ELE treatment, especially in SNAI1 and SNAI2.</p

Additional file 2: Figure S2. of ATF-3/miR-590/GOLPH3 signaling pathway regulates proliferation of breast cancer

Related to Fig. 5. knockdown of ATF-3 inhibited proliferation of MCF-7 cells. (A) MTS assay showed downregulation of ATF-3 by transfecting siRNA inhibits proliferation during 24, 48, 72 h in MCF-7 cells . Data shown are means ± SD (n = 4). *P < 0.05. (TIFF 96 kb

Additional file 1: Figure S1. of ATF-3/miR-590/GOLPH3 signaling pathway regulates proliferation of breast cancer

Related to Fig. 2. Golph3 regulates cell proliferation of MCF-7 cells (A) MTS assay showed promotion of cells proliferation because of Golph3 overexpression during 24, 48, 72 h in MCF-7 cells . Data shown are means ± SD (n = 3). **P < 0.01. (B) BrdU incorporation assay . Data shown are means ± SD (n = 3). *P < 0.05. (TIFF 3999 kb

Additional file 3: Figure S3. of ATF-3/miR-590/GOLPH3 signaling pathway regulates proliferation of breast cancer

Related to Fig. 6. ATF-3/miR-590-3p/Golph3 regulates the proliferation of MCF-7 cells (A) MTS proliferation assay showed overexpression of ATF-3 could rescue miR-590-3p repressing cell proliferation during 24, 48, 72 h in MCF-7. Data shown are means ± SD (n = 3), **P < 0.01. (B) MTS proliferation assay showed knockdown of ATF-3 repressed cell proliferation, which could be rescued by overexpress Golph3 during 24, 48, 72 h in MCF-7. Data shown are means ± SD (n = 3), **P < 0.01. (TIFF 223 kb