Immunofluorescence analysis of LIF was performed using 80% confluent log phase MDA-MB-231 and T-47D, control and PTI treated, cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with LIF Polyclonal Antibody (Product # PA5-79601) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2500 dilution), for 45 minutes at room temperature (Panel a,e,i: Green). Nuclei (Panel b,f,j: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c,g,k: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing no staining for LIF in untreated MDA-MB-231 cells that is enhanced upon PTI treatment (Panel h). Panel l represents T-47D treated with PTI showing no staining for LIF. The images were captured at 60X magnification.