Fig 1.
TGFβ1 induces EMT in certain non-small cell lung cancer cell lines.
(A) 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) Morphology changes of NCI-H1650 and HCC827 cells were not noted after TGFβ1 treatment. (C) 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, ** P<0.01, *** P<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. (D) Western blotting confirmed that TGFβ1 increased Vimentin and reduced E-cadherin protein expression in TGFβ1 sensitive NSCLC line.
Fig 2.
TGFβ1 reduces cell viability and increases cell invasion in TGFβ1 sensitive NSCLC cell lines.
(A) CellTiter-Glo luminescent assays showed that 2.5 ng/ml TGFβ1 treatment for two weeks significantly reduced A549, NCI-H358 and NCI-H1993 cell viability but had no effects on NCI-H1975, NCI-H1650 and HCC827 cell growth (compared to control without treatment, *** P<0.001). (B) Cell invasion assays revealed that 2.5 ng/ml TGFβ1 treatment for two weeks significantly increased the number of A549, NCI-H358 and NCI-H1993 cells which migrated through the chambers. NCI-H1975, NCI-H1650 and HCC827 cells did not exhibit the similar properties.
Fig 3.
TGFβ1 increases the gene expression of NSCLC stem-like cell markers and colony formation ability in TGFβ1 sensitive NSCLC lines.
(A) 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 (** P<0.01, *** P<0.001). (B) Oct4, Nanog and Sox2 expression remained same before and after TGFβ1 treatment. (C) 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 (* P<0.05, ** P<0.01).
Fig 4.
TGFβ pathway is involved in the regulation of VEGF-C expression in TGFβ1 sensitive NSCLC lines.
(A) 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, *** P<0.001). (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. (C) 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. (D) Similarly, siRNA targeting TGFβR1 significantly decreased TGFβ1-induced VEGF-C expression compared to scramble control (*** P<0.001).