Fig. 6

KH-type splicing regulatory protein (KSRP) transcriptionally suppresses neural precursor cell-expressed developmentally downregulated 4 like (NEDD4L) via inducing upregulation of Wilm's tumor 1 (WT1). A Upper panel, schematic representation of the promoter region (1506 bp) of NEDD4L. Lower panel, relative luciferase activities of Caki-1 cells transfected with the NADD4L promoter reporter with or without KSRP shRNA. *** p < 0.001, compared to the control group. B Schematic illustration of truncated NEDD4L promoter constructs (F1–F3). The position of the yellow box represents the fragment location relative to the full-length NEDD4L. C Luciferase reporter activity driven by different NEDD4L promoter segments in Caki-1 cells infected with shKSRP or shCtrl. D Flowchart for identifying possible transcription factors (TFs) involved in KSRP-regulated NEDD4L promoter activity. TF binding to F2 or F3 fragments was predicted by the PROMO and LASAGNA websites. Common TF-binding regions on F2 and F3 were collected, and WT1 was ultimately identified as a candidate TF. E–F WB analysis of KSRP, WT1, and NEDD4L expression levels in whole-cell lysates (E) or cytoplasmic and nuclear fractions (F) from Caki-1 and 786-O cells infected with shKSRP or shCtrl. G-H Luciferase reporter (G) and WB (H) assays were respectively used to detect promoter activities and expression levels of NEDD4L in Caki-1 and 786-O cells infected with shWT1 or shCtrl. I Caki-1 and 786-O cells were infected with shCtrl, shWT1 or shWT1 + shNEDD4L, and the invasive ability of cells were respectively determined by Matrigel invasion assays. J Correlations among KSRP, NEDD4L, and WT1 were analyzed in clear cell renal cell carcinoma (ccRCC) patients from TCGA KIRC and E-MTAB-6692 datasets. Correlation coefficients and p values were evaluated by a Pearson correlation analysis. K Kaplan–Meier analysis of overall survival (OS) rates in patients with ccRCC presenting with high or low expression of WT1 using data from TCGA KIRC dataset