PKCδ inhibition increases Wnt3a-induced cell proliferation and the expression of Wnt target genes in RKO cells.

<p><b>A)</b> Effect of PKCδ knockdown on Wnt target genes expression. RKO cells were transfected with 1 µg of pSuperPKCδ. RNAi or with control plasmid. 28 hours after transfection, cell lysates were prepared to perform Western blot analysis for c-myc and cyclin D1 protein expression (left) or to obtain RNA to perform RT-PCR for c-myc and cyclin D1 mRNA expression (right), respectively. The bar graphs show the quantitative scans of the ratio of c-myc or cyclin D1 protein to the actin content. Values plotted are means ± S.E.M. of at least three independent experiments. *p<0.05 <b>B)</b> Cell proliferation assay. RKO cells were serum starved (2% instead of 10%) during 8 hr and then incubated 24 hr in the absence or presence of 100 ng/ml Wnt3a and in the absence or presence of 6 µM rottlerin. The cells were then treated with 0.5 mg/ml (final concentration) of MTT solution for 3 h at 37°C. Reaction was stopped and the cell samples quantified spectrophotometrically at 570 nm. *p<0.05. <b>C)</b> Stable knockdown of PKCδ increases tumorigenic activity of engrafted RKO cells in nude mice. Stable control or PKCδ knockdown transfectants were obtained as described under Methods sections. Selected clones were picked and screened for PKCδ silencing in comparison with control cells by flow cytometry as shown at the left of the figure. Selected cells were grown (corresponding to 73% PKCδ depleted cells) and used for engraftment. 1 × 10⁶ cells per injection site were subcutaneously injected into the flanks of NOD/SCID nude mice (n = 5). Each mouse was injected at the right flank with control cells, and at the left flank with knockdown PKCδ cells. Five weeks after inoculation, animals were euthanized and tumors were removed and weighted. In all mice, engrafted PKCδ knockdown cells produced bigger tumors in comparison with the engrafted control cells injected in the same animal. The % increase in tumor weight of knockdown cells of each tumor over its respective control was calculated and depicted as the bar graph shown at the left (representing the mean weight increase over controls ± SEM obtained from 5 mice *p<0.05)<b>.</b> At the right, the same data are plotted to show the heterogeneity of the tumors.