TGFβ pathway is involved in the regulation of VEGF-C expression in TGFβ1 sensitive NSCLC lines.

<p><b>(A)</b> Real-time PCR showed that VEGFR3 mRNA expression levels were much higher in A549, NCI-H358 and NCI-H1993 cells than that in NCI-H1975, NCI-H1650 and HCC827 cells (fold expression as compared to control NCI-H1975, *** <i>P</i><0.001). <b>(B)</b> Immunoblotting showed that human recombinant VEGF-C 10 ng/ml treatment for 30 and 60 minutes activated ERK pathway in NCI-H1993 cells but not in NCI-H1975 cells. <b>(C)</b> Real-time PCR revealed that 2.5 ng/ml TGFβ1 treatment significantly increased the VEGF-C mRNA expression in NCI-H1993 cells. The presence of 0.1 μM LY2157299 significantly reduced TGFβ1-induced VEGF-C expression. <b>(D)</b> Similarly, siRNA targeting TGFβR1 significantly decreased TGFβ1-induced VEGF-C expression compared to scramble control (*** P<0.001).</p

TGFβ1 increases the gene expression of NSCLC stem-like cell markers and colony formation ability in TGFβ1 sensitive NSCLC lines.

<p><b>(A)</b> Real-time PCR showed that 2.5 ng/ml TGFβ1 treatment for two weeks significantly increased Oct4 and Sox2 mRNA expression in A549, NCI-H358 and NCI-H1993 cells compared to untreated cells (** <i>P</i><0.01, *** <i>P</i><0.001). <b>(B)</b> Oct4, Nanog and Sox2 expression remained same before and after TGFβ1 treatment. <b>(C)</b> 2.5 ng/ml TGFβ1 treatment for two weeks significantly enhanced anchorage-dependent colony formation ability of A549, NCI-H358 and NCI-H1993 cells but not for NCI-H1975, NCI-H1650 and HCC827 cells (* <i>P</i><0.05, ** <i>P</i><0.01).</p

TGFβ1 induces EMT in certain non-small cell lung cancer cell lines.

<p><b>(A)</b> A549 and NCI-H1993 cell images were taken before and after incubation with 2.5 ng/ml human recombinant TGFβ1 for two weeks. They transited from epithelial to mesenchymal like cells. Bar: 20 μm. <b>(B)</b> Morphology changes of NCI-H1650 and HCC827 cells were not noted after TGFβ1 treatment. <b>(C)</b> Real-time PCR revealed that 2.5 ng/ml TGFβ1 treatment for two weeks significantly reduced E-cadherin mRNA expression in A549 and NCI-H1993 cells (fold expression as compared to control NCI-H1975, ** <i>P</i><0.01, *** <i>P</i><0.001) but not in NCI-H358, NCI-H1975, NCI-H1650 and HCC827 cells. At the same time, Vimentin mRNA expression was significantly enhanced in A549, NCI-H358 and NCI-H1993 cells but not in NCI-H1975, NCI-H1650 and HCC827 cells. <b>(D)</b> Western blotting confirmed that TGFβ1 increased Vimentin and reduced E-cadherin protein expression in TGFβ1 sensitive NSCLC line.</p

Additional file 1: of The characteristics of ctDNA reveal the high complexity in matching the corresponding tumor tissues

Figure S1. UC-Seq significantly improve the sensitivity of mutation detection in ctDNA. (A) Sensitivity of ctDNA detection with or without barcoding. (B) Distribution of mutant allelic frequencies (AFs) in ctDNA with or without barcoding. (PDF 756 kb

Additional file 2: of The characteristics of ctDNA reveal the high complexity in matching the corresponding tumor tissues

Figure S2. The sensitivity of copy gain detection was affected by the copy gain ratio in corresponding tumor tissues. (A) The curve of sensitivity varied with the decreasing of the copy gain ratio cut-offs in corresponding tumor tissues. (B) Scatter plot of maximum MAFs in ctDNA versus copy gain ratio in corresponding tumor tissues. (PDF 868 kb