Fig. 2

MILIP promotes ccRCC metastasis a-e SiRNA knockdown of MILIP (a) inhibited wound healing (b, c) and migration and invasion (d, e) of Caki-1 and ACHN cells. Wound width and area of migrated and invasive cells were quantitated using the ImageJ software. Scale bars, 200 μm (wound healing assay) and 1 mm (cell migration and invasion assay). One-way ANOVA followed by Tukey’s multiple comparisons test. f Gene Set Enrichment Analysis (GSEA) of RNA-seq data from Caki-1 cells with or without MILIP knockdown showing that the metastasis pathway was significantly downregulated after MILIP knockdown. n = 3 experimental repeats. NES, normalized enrichment score. g SiRNA knockdown of MILIP increased E-cadherin levels whereas decreased N-cadherin, MMP2 and Vimentin levels in Caki-1 and ACHN cells. h Overexpression of MILIP caused the decrease of E-cadherin levels and the increase of N-cadherin, MMP2 and Vimentin levels in 769-P cells. i, j Representative green fluorescence (GFP) photographs (i) of lung tissues from NOD/SCID mice injected with ACHN.shMILIP.1 cells via the tail vein. Mice in different groups were treated with doxycycline (Dox, 2 mg/ml supplemented with 10 mg/ml sucrose in drinking water) and PBS, respectively. GFP positive area and whole lung area were quantitated using the ImageJ software (j). Scale bar, 2 mm. n = 8 biologically independent animals; Two-tailed Student’s t-test. k Dox treatment reduced weights of lung tissues from NOD/SCID mice injected with ACHN.shMILIP.1 cells via the tail vein. n = 8; Two-tailed Student’s t-test. l Representative photographs of Hematoxylin and Eosin (H&E) staining of lung tissues from NOD/SCID mice injected with ACHN.shMILIP.1 cells. Scale bar, 0.5 mm. Data are representatives of eight biologically independent animals. Data are representatives or mean ± s.d.; n = 3 independent experiments or 8 biologically independent animals