Fig 6. WNK1 and PRAS40-related pathways play a role in BMCM-mediated migration of breast cancer cells.

(A, B) MDA-MB-231 human breast cancer cells were subjected to siRNA knockdown of WNK1 (A) or PRAS40 (B). Knockdown was confirmed by immunoblotting (top panels) prior to carrying out transwell migration assays (bottom panels; 5 x 10⁴ cells/well; 8μm pore size) using basal media (DMEM/F12 + Mito⁺) or BMCM. (C) MDA-MB-231 human breast cancer cells were serum-starved and treated with 20μM of PI3K inhibitor (LY294002) or Akt inhibitor (Triciribine) or an equivalent concentration of vehicle (DMSO) for 1 hour before being subjected to transwell migration assays (5 x 10⁴ cells/well; 8μm pore size) using basal media (DMEM/F12 + Mito⁺) or BMCM. Plates were incubated at 37°C, 5% CO₂ for 18 hr, fixed, and stained. Five high-powered fields of view (HPF) were captured per transwell and migrated cells were analyzed. Data are presented as mean ± SEM (N = 3; fold change from negative control of basal media). * = significantly different than basal media; ϕ = significantly different than BMCM + siCON (A, B) or BMCM + vehicle (C) (P≤0.05).