Figure 2.

Canine mammary tumor (CMT)-U27 and CMT-U309 cells display differential organization of actin cytoskeleton and expression of actin-associated proteins. (A) Simple carcinoma cells CMT-U27 possess both thick contractile actomyosin bundles as well as thinner actin-based structures both perpendicular and parallel to the leading edge of the cells. Spindle carcinoma cells, CMT-U309, have predominantly thick straight actomyosin bundles that are connected to focal adhesion sites from their both ends. Magnification of the cell edges of both cell lines are shown below. Phalloidin-green, focal adhesion marker vinculin-red and DAPI-blue. Scale 10 μm. (B) Western blot analyses of cellular lysates from the studied cell lines showed that both cell lines express high levels of β-actin, while high expression of smooth muscle actin, α-SMA, was detected only in spindle cell carcinoma U309. These cells were devoid of non-muscle myosin as detected with the phospho-thr18/ser19-myosin light chain (MLC) antibody. Differential expression of calponin-1 and specific tropomyosin isoforms was also detected between these cell lines: CMT-U309 cells expressed high levels of calponin-1, typical for basal cells, while this protein was not detected from the lysates of CMT-U27 cells. CMT-U27 expressed mainly tropomyosin isoform, Tm3, while CMT-U309 cells expressed Tm1 and Tm2. Western Blot experiments were repeated at least three times. (C) Expression of active form of AMP-activated protein kinase (AMPK) kinase, phospho-Thr172-AMPK, was analysed from the cell lines as it has been linked to maturation of thick contractile actomyosin bundles (26). High levels of P-Thr172-AMPK was detected from the lysates of CMT-U309 cells, correlating with the appearance of thick actomyosin bundles in these cells.