Figure 2

Effects of PKCδ inhibitors on human cancer stem cells. (A) PCSC and PrCSC cells at 80% confluence were exposed to rottlerin. DMSO served as vehicle control (Vehicle). After 24 and 72 hr of exposure, cell mass was evaluated by MTT assay. Control values were normalized to 100%. p values for comparison between treatments reached significance at 24 hr of exposure (p≤0.01) for both cell types, and remained significant at 72 hr. (B) PCSC and PrCSC cells at 50% confluence were exposed to rottlerin. Cytotoxicity was evaluated by LDH-release assay. Total maximal LDH release was assigned the arbitrary value of 100% (Control). p values for comparison between effects of treatments on LDH release reached significance at 24 hr of exposure (p<0.01) for both cell types, and remained significant at 72 hr. (C) Effects of PKCδ inhibitor on tumor cell clonogenic capacity. PCSC were exposed to vehicle or rottlerin (10 μM) for 6, 18, 24, or 48 hr. Viable cells were enumerated and re-plated in media without inhibitor, and colony numbers were quantitated 15 days later. p values for comparison of treatment effects on clonogenic capacity reached significance (p=0.005) at 18 hr of exposure and remained significant for all subsequent exposure times. The insert is a photograph of stained colonies on plates. (D) Structures of staurosporine, rottlerin, second-generation (KAM1) and third-generation (BJE6-106) derivatives. (E) PCSC and PrCSC cells at 50% confluence were exposed to KAM1 at the indicated concentrations. DMSO served as vehicle control (Vehicle). Cytotoxicity was evaluated by LDH-release assay, as in panel B. p values for comparison between treatment effects on LDH release reached significance at 24 hr of exposure to 2.5 μM KAM1 for PCSC cells and at 10 μM for PrCSC (p≤0.01), and remained significant at 72 hr for all concentrations of KAM1. Error bars represent SEM.