Bet1 is required for efficient ECM degradation. (A) Screening of SNARE proteins involved in invadopodium-mediated gelatin degradation by overexpression of dominant-negative SNAREs. MDA-MB-231 cells transiently expressing FLAG-tagged, TMD-deleted SNAREs were cultured on TRITC-gelatin for 7 h, fixed, and stained for FLAG or cortactin, and then the cells exhibiting gelatin degradation were counted under a fluorescence microscope. (B and C) siRNA-mediated knockdown of Bet1 reduces gelatin degradation. MDA-MB-231 cells transfected with the indicated siRNAs were subjected to gelatin degradation assaying as in A. Representative pictures of gelatin degradation (B) and quantitation of the ratio of the cells exhibiting gelatin degradation (C) are shown. (D) WT Bet1, but not its ΔSNARE mutant, rescues the reduced gelatin degradation with Bet1 knockdown. MDA-MB-231 cells stably expressing WT Bet1-GFP or its ΔSNARE mutant were transfected with Bet1 siRNA#3, which targets the 3′-UTR of Bet1 mRNA and depletes endogenous Bet1, but not Bet1-GFP or its ΔSNARE mutant, and then gelatin degradation was examined as in A. (E and F) CRISPR-mediated KO of Bet1 genes abolishes gelatin degradation. MDA-MB-231 cells in which the Bet1 gene was disrupted were subjected to gelatin degradation assaying as in A. Representative pictures of gelatin degradation (E) and quantitation of the ratio of cells exhibiting gelatin degradation (F) in the Bet1-KO cells are shown. (G) Bet1 knockdown impairs invasion through a Matrigel matrix. Invasion activity of Bet1-depleted MDA-MB-231 cells was assessed by Matrigel-coated Transwell assay using 10% FBS as an attractant. Scale bar: 10 μm. *, P < 0.05; **, P < 0.01; vs. mock in C; vs. parental MDA, mock in D; vs. parental MDA in F; vs. mock (+ serum) in G.