Fig. 1

Suppressive role of FBXO9 in migration, tumor sphere formation and metastasis of lung cancer cells. A Schematic diagram illustrating the role of F-box proteins in CRL1/SCF ubiquitin ligase. B Fbxo9 expression, including that of the Fbxo9-ΔF mutant lacking the F-box domain, is controlled using the Tet-On system. C Co-immunoprecipitation assay identifying the binding capacity between Fbxo9-ΔF and the Skp protein. D Immunoblot analysis of 889DTC cells expressing Fbxo9 and Fbxo9-ΔF, with Gapdh as a loading control. E Representative images and quantitative results of migrated 889DTC cells expressing either Fbxo9 or Fbxo9-ΔF(P < 0.001). F Immunoblot analysis of H1299 cells expressing FBXO9 and FBXO9-ΔF, with GAPDH serving as a loading control. G Representative images (left) and quantitative results (right) of migrated H1299 cells expressing FBXO9 or FBXO9-ΔF (P < 0.0001). H Immunoblot analysis of Fbxo9 knockdown in 889DTC cells using shRNA, with Gapdh as a loading control. I Representative images (left) and quantitative results (right) of migrated 889DTC cells with Fbxo9 knockdown (P < 0.01). J Immunoblot analysis confirming FBXO9 knockdown in H1299 cells using shRNA. K Representative images (left) and quantitative results (right) of cell migration in H1299 cells with FBXO9 depletion (P < 0.0001). L Impact of Fbxo9 knockdown on tumor sphere formation in 889DTC cells (P < 0.0001). M Impact of FBXO9 knockdown on tumor sphere formation in H1299 cells (P < 0.0001). N Ectopic expression of Fbxo9 reduces lung cancer cell metastasis in vivo. Nude mice were injected with 889DTC cells expressing Fbxo9 via the tail vein (n = 6 each group). After two weeks, lung tissues were analyzed. Fluorescence microscopy detected the presence of 889DTC-Tdtomato cells, as indicated by orange fluorescence (left). The number of metastasis nodules was quantified based on fluorescence and statistically analyzed (right) (P < 0.001). O Downregulation of FBXO9 promotes lung cancer cell metastasis in vivo. H1299-Tdtomato cells with stable FBXO9 knockdown were injected into the tail vein of nude mice (n = 6 each group). After 6 weeks, lung tissues were collected for fluorescence microscopy (left) and H&E staining (left). Metastasis nodules were quantified via fluorescence microscopy and statistically analyzed (right) (P < 0.01). All error bars represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001