Fig 2: Silencing of INAVA inhibits cell invasion, migration and metastasis. a Protein expression levels of INAVA in INAVA-silencing and vector-control cells were analyzed by WB. α-Tubulin was used as a loading control. Representative images (b) and quantification (c) of transwell migration assays in indicated cells. Representative images (d) and quantification (e) of transwell invasion assays in indicated cells. f Scratch wound healing assays were performed in indicated cells (left) and quantification analyses for the assays were shown (right). g Representative bioluminescence images in mice with tail vein injection of the indicated cells (left) and representative histopathology of lung metastasis stained with HE was shown (right). For b–f, data are quantified as mean ± SD of 3 independent experiments in the bar graphs. *P < 0.05

Fig 3: INAVA modulates MMP9 via regulating FGF1. mRNA levels of FGF1 were examined in INAVA-overexpressing (a) or -knockdown cells (b). c, d Protein levels of FGF1 were assessed in indicated cells. e ELISA assays were performed to assess the effect of INAVA on FGF1 release in TPC1 and K1 cells. f Knockdown of FGF1 by siRNAs in INAVA-overexpressing cells reversed increased level of MMP9. g Invasiveness caused by INAVA was abrogated by silencing FGF1 expression in INAVA-overexpressing cells. For a, b, e, g, results derived from three independent experiments are expressed as mean ± SD. *P < 0.05

Fig 4: INAVA positively correlates with FGF1 and MMP9 in PTC clinical specimens. a The expression of INAVA is associated with FGF1 and MMP9 in clinical PTC specimens. Two representative cases are shown. b Percentage of cases showing low (n = 57) or high (n = 55) INAVA expression in relation to the FGF1 and MMP9 expression levels. c The association between INAVA and FGF1 as well as MMP9 in 496 cases of PTC specimens using RNAseq data deposited on TCGA. The expression level of INAVA was divided into low (n = 248) and high (n = 248) groups according to median distribution. For b, c, *P < 0.05

Fig 5: INAVA is upregulated in PTC and is associated with LN metastasis. a The expression of INAVA in 59 pairs of primary PTC (T) versus their paired non-cancerous thyroid tissues (N) using RNAseq data sets deposited on TCGA. b The expression of INAVA in 16 paired T and N was assessed by real time RT-PCR. GAPDH was used as a house keeping gene control. c Representative images of IHC assays on INAVA expression in PTC specimens from the studied cohort. NLN with no lymph node metastasis, LN with lymph node metastasis. d Percentage of specimens showing low (n = 57) or high (n = 55) INAVA expression in relation to the LN metastasis. *P < 0.05