Figure 4

Effects of PKCδ inhibitors on human tumor cell spheroid formation. (A) Hs578T and MCF7 were plated under adherent or non-adherent conditions. Tumor spheroids and adherent cells were collected at 96 hr, stained for CD24 and CD44, and analyzed by flow cytometry. (B) Hs578T, MCF7, breast cancer stem cells (BCSC) and pancreatic cancer stem cells (PCSC) were plated in tumor spheroid media, in the presence of rottlerin, BJE6-106, or DMSO (Control). Tumor spheroids were enumerated at 96 hr, and normalized to the number of spheroids in the control cultures (assigned an arbitrary value of 100%). p values for comparison between vehicle and rottlerin or BJE6-106 effects were significant (p≤0.001). Photographs are of representative areas of the culture plates. (C) MCF7 cells were exposed BJE6-106 or to rottlerin at the indicated concentrations. 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 values were normalized to 100%. p values for comparison between vehicle and rottlerin effects on cell number at 24 hr reached significance at 5 μM, and for BJE6-106 at 0.5 μM (p ≤ 0.02), and were significant for all concentrations tested at 48 and 72 hr time points. (D) Hs578T cells were exposed to vehicle or BJE6-106 (1 μM) for 6, 12, 24, 48 or 96 hr. Viable cells were enumerated and re-plated in media without BJE6-206, and spheroid numbers were quantitated 96 hr later. p values for comparison between vehicle and BJE6-106 effects on spheroid number were significant after 6 hr of exposure (p≤0.02), and remained significant at all time points thereafter. Error bars represent SEM.