Fig. 1

MILIP is expressed at higher levels in metastatic ccRCC and is transcriptionally activated by TFAP2C a, b MILIP was expressed at higher levels in metastatic compared with primary ccRCC tissues and its high level was associated with poor patient prognosis as revealed by analysis of the transcriptome array data from a GEO dataset (GSE22541). Two-tailed Student’s t-test (a). The log rank test (b). c Analysis of RNA-seq data of Cancer Cell Line Encyclopedia (CCLE) dataset showing that MILIP was expressed at higher levels in metastatic compared with primary ccRCC cell lines. Two-tailed Student’s t-test. d, e Representative microphotographs (d) and quantitation (e) of in situ hybridization (ISH) analysis of MILIP expression in metastatic compared with primary ccRCC tissues. The red arrows indicate MILIP. Scale bar, 20 µm. RS: reactive score. Two-tailed Student’s t-test. f MILIP was expressed at higher levels in metastatic than primary ccRCC cell lines. One-way ANOVA followed by Tukey’s multiple comparisons test. g A schematic illustration of a consensus TFAP2C binding region (TFAP2C-BR) and its ChIP-seq peak in MILIP promoter. h ChIP-PCR analysis of the association between endogenous TFAP2C and the TFAP2C-BR. i, j Overexpression of TFAP2C increased whereas knockdown of TFAP2C decreased the transcriptional activity of a MILIP reporter construct with the intact TFAP2C-BR (pGL3-MILIP-promoter) but not with the TFAP2C-BR deleted (pGL3-MILIP-promoter-ΔBR). FL: firefly luciferase activity; RL: renilla luciferase activity. One-way ANOVA followed by Tukey’s multiple comparisons test. k Silencing TFAP2C downregulated MILIP expression. One-way ANOVA followed by Tukey’s multiple comparisons test. l Kaplan-Meier analysis showing that high TFAP2C level was associated with poor patient prognosis using median of TFAP2C level as the cutoff. The log rank test. m, n qPCR (m) and ISH (n) analysis showing that MILIP was predominantly localized in the cytoplasm. GAPDH: cytoplasmic marker; Neat1: nuclear marker; DapB: negative control; PPIB: positive control. Scale bar, 10 µm. Data are representatives or mean ± s.d.; n = 3 independent experiments