Fig. 7

RANK-induced canonical and noncanonical NF-κB pathways have distinct roles in the pregnancy-dependent expansion of mammary epithelial cells. a Western blotting analysis of p-IκBα and IκBα. NMuMG-RANK clone#1 (WT) and #4 (KO) were pretreated with serum-reduced DMEM (1% FBS) for 8 h and then stimulated with rRANKL. b Western blotting analysis of p100, p52, and Cyclin D1 expression. NMuMG-RANK clones were pretreated as in a and then stimulated with rRANKL for 24 h. c Real-time RT-qPCR analysis of Birc3, Tnfaip3, and Ccnd1 expression in NMuMG-RANK clones. Total RNA was prepared and subjected to RT-qPCR. Values are means ± SD (n = 3; **p < 0.01). d, e Effect of TPCA-1, an IKKβ inhibitor, on canonical (d) and noncanonical (e) pathways. NMuMG-RANK parent cells were pretreated as in a and then treated with TPCA-1 alone for 30 min followed by stimulation with GST-RANKL with TPCA-1. Whole-cell lysates were prepared and subjected to western blotting analysis. f Real-time RT-qPCR analysis of Birc3, Tnfaip3, and Ccnd1. NMuMG-RANK parent cells were pretreated as in a and then treated with TPCA-1 alone for 30 min followed by GST-RANKL stimulation with TPCA-1. Total RNA was prepared and subjected to RT-qPCR. Values are mean ± SD (n = 3; **p < 0.01). g, h Effect of NIK-knockdown on canonical (g) and noncanonical (h) pathways. NMuMG-RANK parent cells were transfected with control siRNA or two distinct siRNAs against Map3k14 (NIK, #1 and #2) for 24 h. Cells were then pretreated as in a followed by GST-RANKL stimulation. Whole-cell lysates were prepared and subjected to western blotting analysis. i Real-time RT-qPCR analysis of Birc3, Tnfaip3, and Ccnd1 expression. Control or NIK siRNA-treated NMuMG-RANK cells were pretreated as in a and then stimulated with GST-RANKL. Total RNA was prepared and subjected to RT-qPCR. Values are means ± SD (n = 3, **p < 0.01)