Fig. 4

FBXO9-mediated ATP6V1A ubiquitination impairs metastasis of lung cancer cells. A, B Ubiquitination assays were performed in HEK293T cells by co-transfection with full-length HA-ATP6V1A, HA-ATP6V1A-KR (lysine residues removed), HA-ATP6V1A-K393 (K393 reintroduced into the ATP6V1A-KR mutant), or HA-ATP6V1A-K393R (single lysine substitution mutant), in the presence or absence of FLAG-FBXO9. After 48 h, cells were lysed, followed by Ni-NTA bead pull-down and immunoblotting for HA-ATP6V1A. C Alignment of ATP6V1A K393 conservation across species. D Ubiquitination analysis of K393 in HA-Atp6v1a by co-expressing Flag-Fbxo9 and His-Ub with indicated HA-Atp6v1a constructs (WT, K393, or K393R) in HEK293T cells. Cells were lysed after 48 h, followed by Ni-NTA bead pull-down and immunoblotting for HA-Atp6v1a. E V-ATPase complex reconstitution was achieved in H1299 cells by introducing sgRNA-resistant ATP6V1A (wild-type or K393R mutant) using a Tet-Off system. CRISPR was then used to deplete endogenous ATP6V1A, allowing the V-ATPase complex to be reconstituted. Immunoblot analysis confirmed comparable expression levels of the added ATP6V1A and endogenous subunits ATP6V1B and ATP6V1D1, in the experimental group compared to the control cells, after 48 h of doxycycline treatment. F–I Recombinant cell lines H1299-KO-V1A-wt, H1299-KO-V1A-K393R, and H1299-EGFP-sgCtrl were used in a transwell migration assay (F, G) and tumor sphere formation assay (H, I) to evaluate the effects of ATP6V1A ubiquitination on cell migration and tumor sphere growth(P < 0.001). J–N To assess the effects of ATP6V1A ubiquitination on cell migration and tumor sphere growth, V1A-23aa was introduced into A549 cells to inhibit ATP6V1A ubiquitination (J) which was followed by transwell migration assay (K, L) and tumor sphere formation assay (M, N) to evaluate the impact on cell migration and tumor sphere growth, respectively (P < 0.001). O, P Impact of ATP6V1A ubiquitination inhibition by FBXO9 on in vivo lung metastasis was assessed. A549 cells expressing V1A-23 aa were injected into mice via the tail vein. After 6 weeks, the lungs of the mice were harvested for examination and H&E staining. Metastasis nodules were quantified and statistically analyzed using fluorescence microscopy. Error bars represent the mean ± SD. ****P < 0.0001 indicates the significance level