Figure 5

Effects of PKCδ inhibitors on growth and spheroid formation in non-transformed and transformed human breast epithelial cells. MCF 10A cells and cells from the derived tumorigenic line MCF 10C (also called M3), were grown to 80% confluence in 96-well plates and then exposed to rottlerin at concentrations ranging from 1 to 20 μM (A) or to BJE6-106 at concentrations ranging from 0.1 to 20 μM (B). The corresponding equivalent volume of solvent (DMSO) was used as a vehicle control (Vehicle). After 24, 48 and 72 hr of exposure, cell mass was evaluated by MTT assay. Control (vehicle) values were normalized to 100%. Error bars represent SEM. p values for comparison between vehicle and PKCδ inhibitors on MCF 10A cell number only reached significance (p < 0.05) at 48 hr at 20 μM for rottlerin, and at 1 μM for BJE6-106. In contrast, significant effects of the inhibitors on the MCF 10C cells were observed as early as 24 hr for rottlerin (at 5 μM) and for BJE6-106 (at 0.1 μM). (C) MCF 10A and MCF 10C cells were plated at 10,000 cells per well in tumor spheroid media, and spheroid formation was assessed at days 10 and 21. Representative photographs are shown. (D) MCF 10C cells were plated at 10,000 cells per well in tumor spheroid media, in the presence of rottlerin (5 μM), or BJE6-106 (1 μM or 5 μM), or DMSO vehicle (Control). Tumor spheroids were enumerated at 10 days. Representative photographs are shown. (E) Spheroid numbers were normalized to the number of spheroids in the control cultures (assigned an arbitrary value of 100%) and plotted. Error bars represent SEM. p values for comparison between vehicle and rottlerin or BJE6-106 effects on spheroid number were significant (p < 0.001).