Fig. 6

POSTN increases expression of TGF-β2 capable of inducing CAF markers and is correlated with abundant CAFs in tumor microenvironment. A The qRT-PCR analysis of α-SMA and FAP mRNAs from conditioned medium treated hADSCs. The conditioned media were collected from SKOV3/ctrl or SKOV3/POSTN cells cultured for 2 days. B MTT proliferation assay of SKOV3 cells incubated with conditioned medium from unprimed, SKOV3/ctrl cells or SKOV3/POSTN cells primed hADSC cells. C, D RNA and protein levels of TGF-β1 and TGF-β2 in POSTN-overexpressing SKOV3 cells were determined qRT-PCR and ELISA, respectively. E Expression levels of α-SMA and FAP transcripts in TGF-β2 treated normal human adipose derived stromal cells (hADSCs) were determined by qRT-PCR. F Representative immunohistochemical analysis for the expression of α-SMA in POSTN-overexpressing versus control SKOV3-derived primary and metastatic tumors in the orthotopic metastasis mouse model (left panel). Quantification results of the immunohistochemical analysis of anti-α-SMA (right panel). Six female NOD/SCID mice were randomly assigned to each group and were followed up for 3 weeks in orthotopic metastasis mouse model. G Western blot analysis for measuring the POSTN level in SKOV-I6 cells infected with lentiviral plasmid encoding shPOSTN or a scrambled control. H The diagram illustrates the co-culture system used for cancer cell-induced CAF markers activation. I Representative immunofluorescence images showe the α-SMA levels of hADSC in non-co-cultured (left), co-cultured with SKOV-I6/SC (middle) or co-cultured with SKOV-I6/shPOSTN#1 cells (right). Scale bar = 50 μm. J Western blot analysis for detecting TGFB2 and POSTN in POSTN overexpressing SKOV3 cells infected with lentiviral vector shTGFB2 or a scrambled control. K The diagram illustrates the co-culture system used for cancer cell-induced CAF markers activation. L Representative immunofluorescence images showing the α-SMA levels of hADSC in co-cultured with SKOV3/ctrl (left), SKOV3/POSTN-scrambled control (middle) or SKOV3/POSTN-shTGFB2#2 cells (right). *p < 0.05; **p < 0.01; ***p < 0.001