Inhibition of the NFκB pathway on HA-BrC cells

To inhibit the NFκB pathway in HA-BrC cells HS578T and MDA-MB-231, we used the selective IKKα and IKKβ inhibitor ACHP from Tocris Bio-techne brand (REF 4547) [67]. ACHP was prepared as a 20 mM stock solution in dimethyl sulfoxide (DMSO) and stored at −70° C until use. 2 × 10⁶ cells of HS578T and MDA-MB-231 were plated in 182 cm² flasks in their standard supplemented medium, supernatants were discarded 24 hours later and cells were rinsed with PBS 1×, adding 30 mL of their respective culture media without FBS plus 16 nM/mL of ACHP. After 48 hours of culture, CMs were harvested, centrifuged at 1500 rpm/5 minutes, aliquoted, and stored at −20° C until use. As controls, we obtained CMs from cells cultures without ACHP. In order to generate a transient defective signal from the canonical NFkB pathway, we also transfected Hs578T and MDA-MB-231 cells with a plasmid vector expressing a dominant-negative IκB (mutated at serine 32 and 36) [68]. 24 hours after transfection, cells were washed with PBS 1×, after which we added their respective culture media without FBS. After 48 hours of culture, CMs were harvested, centrifuged at 1500 rpm/5 minutes, aliquoted, and stored at −20° C until use. MCF-7 and T47D cells were cultured with 3 mL of CM of HA-BrC cells cultured with ACHP or 3 mL of CM of HA-BrC cells transfected with the dominant negative IκB mutant. After incubation for 72 hours, MCF-7 and T47D cells were harvested and the induced invasive/stemness phenotype was evaluated.