Figure 5.

CDYL2 Regulation of NF-κB and STAT3 Signaling Contributes to Its Induction of Invasion and Mammosphere Formation

(A) Selected gene expression signatures enriched in the indicated RNA-seq datasets.

(B) Western blot of Ser536-phosphorylated p65 and Tyr705-phosphorylated STAT3 in MCF7-Vector versus MCF7-CDYL2. The levels of total p65, STAT3, and β-actin were also probed.

(C) As for (B), except comparing MDA-MB-231 cells treated with esiLuc or esiCDYL2.

(D and E) Western blot validation of RNAi knockdown of p65 (D) or STAT3 (E) in MCF7-Vector and MCF7-CDYL2 cells. β-actin is shown as loading control.

(F) qRT-PCR analysis of the effect of RNAi knockdown of p65 on the expression of a panel of NF-κB target genes that were up-regulated in MCF7-CDYL2 compared with MCF7-Vector cells. Data are represented as mean of three independent experiments ± SD. All differences were significant at p < 0.05 (t test).

(G) As in (F), except the effect of RNAi knockdown of STAT3 on the expression of a panel of its target genes up-regulated in MCF7-CDYL2 compared with MCF7-Vector cells was evaluated.

(H and I) xCELLigence invasion assays of MCF7-CDYL2 in MCF7 cells treated with either control RNAi or siRNA targeting p65 (H) or STAT3 (I). Graphs are representative of three independent experiments in quadruple runs per condition. Error bars represent the SD of quadruplicate readings at each time point.

(J and K) Mammosphere assay of MCF7-CDYL2 in MCF7 cells treated with either control RNAi or siRNA targeting p65 (J) or STAT3 (K). Mammospheres from 1,000 seeded cells with size >50 μm were counted after 8 days. Shown is a scatterplot of the results of a representative of three independent experiments indicating the median (black bar) and t test significance (∗∗∗p < 0.001; ∗∗∗∗p < 0,0001; ns, not significant).