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 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

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