Fig. 8

FOXF2 enhances the osteolytic bone metastasis of breast cancer cells by transactivating CTSK expression. a Schematic of the candidate FOXF2-binding sites in the CTSK promoter region (left) and the binding of FOXF2 to the CTSK promoter region containing the candidate-binding sites in MDA-MB-231 cells transfected with pcDNA3.1-FOXF2-Flag was determined by ChIP assays using anti-Flag antibody or IgG control (right). b The transcriptional activity of the CTSK promoter in the indicated cells was assessed by dual-luciferase reporter assays. c CTSK mRNA (left) and intracellular protein (right) levels in MDA-MB-231 cells, treated as indicated, were detected by RT-qPCR and immunoblot. d CTSK secretion levels in CM from MDA-MB-231 cells, treated as indicated, were detected by immunoblot. e CTSK protein expression in bone metastatic tissues in mice-bearing MDA-MB-231 cells treated as indicated was detected by immunohistochemistry. Arrows point to CTSK + cells. f The bone-matrix degradation and invasive capacity of the indicated cells were assessed by transwell assays. The insert was coated with bone matrix generated by MC3T3E1 cells. g The ability of cancer cells to induce osteoclast differentiation was assessed by incubation of pre-induced primary preosteoclasts with cancer cell CM containing 50 ng ml^–1 RANKL. The number and size of mature TRAP-positive multinucleated (≥3 nuclei) osteoclasts were analyzed. Scale bars: 100 μm. *P < 0.05 by Student’s t test. Error bars are defined as s.d. h CTSK expression levels in metastatic tissues in bone (n = 18) and in other distant organs (n = 47) were compared based on the GSE14020 data set. i Kaplan–Meier analysis of BOMFS of patients with different CTSK (left) or combined FOXF2 and CTSK (right) mRNA levels in primary breast cancers was performed based on the GSE2034_GSE2603 data set. j CTSK and phospho-SMAD1/5 protein levels in MDA-MB-231 cells, treated as indicated, were detected by immunoblot