A 11002

Antibodies were as follows: mouse anti-β-actin (AC-15) (A5441, Sigma-Aldrich, WB 1:2,000), mouse anti-CLC (CON.1) (ab24579, Sigma-Aldrich, WB 1:1,000), mouse anti-CHC (X-22; ABR) (MA1-065, Invitrogen, IF 1:200), goat anti-ARP2 (N-15) (sc-10125, Santa Cruz Biotechnology, WB 1:500), goat anti-ARP3 (G-15) (sc-10130, Santa Cruz Biotechnology, WB 1:500), goat anti-p34-ARPC2 (IMG-3497, Imgenex, WB 1:500), mouse anti-p21-ARPC3 (ab96137, Transduction Laboratories, WB 1:500), rabbit anti-RFP polyclonal (AB3216, Chemicon, IF 1:200), mouse anti-vinculin (ab18058, Abcam, WB 1:1,000), rabbit anti-GFP polyclonal was a kind gift from G. Kops, mouse anti-EGFR (Ab-1 528, Calbiochem, IF 1:50), rabbit anti-AKT (C67E7, Cell Signaling, WB 1:1,000), rabbit anti-pAKT (Ser473) (D9E, Cell Signaling, WB 1:1,000), rabbit anti-p44/42 ERK (20G11, Cell Signaling, WB 1:1,000) and anti-ERK (9102, Cell Signaling, WB 1:1,000), rabbit anti-Integrin β5 (3629, Cell Signaling, IF 1:1,000) and rabbit anti-N-WASP (30D10, Cell Signaling, WB 1:1,000). Alexa-fluor-532 and Alexa-fluor-647 conjugated goat anti-mouse and anti-rabbit IgG (H+L) antibodies (A-11002, A-11009, A-32728, A-32733, Invitrogen, 1:200 dilution). Alexa-fluor-647 phalloidin (A22287, Invitrogen, 1:50 dilution).

Cells were grown on 22-mm-glass coverslips in 6-well plates to ~80% confluency before exposure to appropriate drugs or vehicular controls. After washing with PBS, cells were fixed with 4% formaldehyde in PBS at room temperature (RT) for 10 min. Cells were permeabilized with 0.1% Triton X-100 for 10 min, then incubated with blocking medium (PBS, 0.1% Tween-20, 10% normal goat serum) for 30 min in a humidified chamber at RT. Blocked samples were then stained with mouse anti-Kaiso (Abcam, ab12723; 1:1000) and rabbit anti-LC3A/B (Abcam, ab128025; 1:1000) in antibody diluent (PBS, 0.1% Tween-20, 1% BSA) for 1 h at RT in a humidified chamber. Anti-LC3A/B antibody recognizes both LC3A and LC3B proteins. After appropriate PBS washing, cells were stained with anti-mouse Alexa Fluor 488 (Invitrogen, A-11001; 1:2000) and anti-rabbit Alexa Fluor 594 (Invitrogen, A-11002; 1:1000) secondary antibodies in a humidity chamber in the dark for 1 h at RT. Coverslips were washed and then mounted on slides using ProLong Gold antifade reagent (Molecular Probes, P36934). MDA-MB-231 and MCF-7 used in this study were obtained from ATCC and identify was validated by STRS profiling. All cells were tested and found to be free of mycobacterial contamination.