Figure 4: Mechanisms underlying SFK activation leading to adaptive resistance in DTCs.

(a) Graph shows quantification of activated SFK in MDA-MB231 treated with high concentration docetaxel post siRNA knockdown of CD44, CD24 or both genes in (N=4, ANOVA **P<0.01). (b) Hck was immunoprecipitated from DTCs or parent MDA-MB-231s cell lysates followed by western blotting for CD44 and CD24 antibodies IgG input included for control. IgG HC indicates heavy chain (HC) bands. (c) Co-IP of Hck was performed from cell lysate of parent or DTCs generated from MDA-MB-231 cells transfected with siRNA targeting CD24 and CD44 or a combination of both. Western blotting indicates Cav-1 scaffolding to Hck. (d) Representative confocal images demonstrates colocalization of CD44, CD24 and Hck to lipid raft-rich regions of the cell membrane in DTC derived from MDA-MB-231 cells. Scale bar, 5 μm (e) Confocal microscopy identifies Caveolin 1 (Cav-1) colocalizing with CD24, CD44 and Hck in the MDA-MB-231 DTCs. Scale bar, 5 μm (f) Subcellular localization of Hck in DTC generated following siRNA-knockdown Cav-1. β-Actin and PARP indicate loading controls of cytoplasmic and nuclear compartments, respectively. (g) Confocal microscopy was used to identify subcellular localization in the nuclear plane of phosphorylated Hck (pHck) in the MDA-MB-231 parent compared with DTCs (upper panel). Dual staining shows a pattern of nuclear and perinuclear localized pHck and Cav-1 in the DTCs. Scale bar, 8 μm. (h) Representative confocal images reveal the expression of APAF-1 (Green signal) in MDA-MB-231 DTCs treated with vehicle or RK20449 (1 μM) for 24 h, and counterstained with DAPI. Scale bar, 8 μm.