Fig. 4: LPXN serves as the critical effector of HLF mediating lung metastasis.

a Integrated analyses of HA-tagged HLF ChIP-seq and RNA-seq data. Coordinated DEGs (1144 genes) from RNA-seq data of both HLF overexpression (OE) and depletion (KO) were narrowed down to 258 genes using a Log2FC cut-off of ±0.5, by overlapping with HLF top 10k peak-associated DEGs (2855 genes) from HLF-HA ChIP-seq data, and cell motility-related genes (134 genes) were identified. Potential critical target genes are indicated on the right. n = 3 biological replicates for RNA-seq, n = 2 technical replicates for ChIP-seq. b Representative images and quantification of transwell invasion assay and 3D collagen invasion assay in 786-O cells transduced with sgCtrl or sgHLF followed by infection with lentivirus expressing pLX304-empty vector (Ctrl_EV) or HLF-targeted genes in pLX304 backbone, n = 6 independent cell cultures. Scale bar, transwell invasion, 100 µm; 3D spheroid invasion in collagen, 200 µm. c Representative images of primary tumors (PT) and lung metastatic tumors (Lung-met), and signals of Lung-Met relative to its matching PT from luciferase-labeled 786-O cells transduced with sgCtrl or sgHLF followed by infection with lentivirus expressing pLX304-empty vector (Ctrl_EV) or pLX304-LPXN and orthotopic injection into the renal sub-capsule of NSG mice, n = 5 mice in each group. d Immunoblotting analysis of the indicated ccRCC cell lines following HLF overexpression or knockout. Each experiment has been repeated at least two times with similar results. For the left panel, the samples derive from the same experiment but different gels for LPXN, Vinculin and another for V5 were processed in parallel. For the right panel, the samples derive from the same experiment but different gels for LPXN, Vinculin, another for FLAG and another for V5 were processed in parallel. e ChIP-seq binding peaks of HA-tagged HLF (exogenous), H3K4me3, and H3K27ac at the promoter region of LPXN. f LPXN promoter reporter assay and quantification in 786-O cells overexpressed with empty vector (Ctrl_EV) or pLX304-V5-HLF (HLF) followed by infection with lentivirus expressing pEZX-LvPF02-LPXN promoter-eGFP. 40x magnification, scale bar, 50 µm, n = 7 independent cell cultures. Data are mean ± s.e.m. (f), box plots show the median and interquartile range, and whiskers show the data range (b, c), One-way ANOVA followed by a post hoc Dunnett-t-test (b, c) or unpaired two-tailed t-test (f). Source data are provided as a Source Data file.