Figure 6. NF-κB Controls Master TFs of MES Differentiation in GSCs.

(A) Western blot analysis of phosphorylated p65 (ser 536), total p65, phosphorylated STAT3 (Tyr 705), STAT3 and C/EBPβ in GSCs. (B) and (C) Western blot analysis using indicated antibodies were performed on GSC23 and 11 sorted for CD44^high or CD44^low subpopulations. (D) Box plots of normalized expression of STAT3, CEBPB, TAZ, and NF-κB metagene in CD44^low (green boxes) or CD44^high (red boxes) tumors from multiple datasets as indicated. Boxes show median 25^th and 75^th percentile, while whiskers represent the 5^th and the 95^th percentile. Outliers are shown as individual points. p value was determined using a non-parametric Wilcoxon test. For the NF-κB metagene, the average expression of 38 NF-κB family members and targets (see Supplemental Experimental Procedures) was condensed into a metagene and plotted. Wilcoxon signed-rank test was used to test statistical significance. (E) Time course western blot analysis of indicated antibodies after TNFα treatment in GSC11 transduced with RFP or IκB-SR adenovirus 24 h prior to TNFα treatment. (F) qRT-PCR analysis of MES signature master TFs STAT3, CEBPB and TAZ in GSC11 treated with TNFα with or without pre-treatment with RFP or IκBSR adenovirus. Error bar indicates +/− SD. t test was used for statistical significance. *p < 0.05 and **p < 0.005. (G) qRT-PCR analysis of YKL40, and CD44 after knockdown of all three master TFs (STAT3, C/EBPβ and TAZ) in GSC11 is shown. Cells were treated with siRNA 72 h prior to treatment with TNFα for an additional 24 h. Error bar indicates +/− SD. t test was used for statistical significance. *p < 0.05 and **p < 0.005. See also Figure S6.