Fig. 5
From: Myeloid-derived interleukin-1β drives oncogenic KRAS-NF-κΒ addiction in malignant pleural effusion
IL-1β-induced NF-κB signaling of KRAS-mutant cells is IKKα dependent. Five different C57BL/6 mouse tumor cell lines with (LLC, MC38, AE17) or without (B16F10, PANO2) Kras mutations were stably transfected with pNGL NF-κΒ reporter and any of the following: control shRNA (shC) or shRNA targeting IKKα (shChuk), ΙΚΚβ (shIkbkb), ΙΚΚε (shIkbke), or TBK1 (shTbk1) transcripts; control plasmid (pC); or overexpression vectors encodong IKKα (pChuk), ΙΚΚβ (pIkbkb), ΙΚΚε (pIkbke), or TBK1 (pTbk1) transcripts. a Immunoblot of cytoplasmic protein extracts from LLC cells stably expressing shC, shChuk, or shIkbkb for IKKα and IKKβ relative to β-actin (representative of n = 3 independent experiments). b Bioluminescent quantification of NF-κΒ reporter activity of pNGL cell lines stably expressing shC, shChuk, shIkbkb, shIkbke, and shTbk1, (n = 3 independent experiments). c Bioluminescent quantification of NF-κΒ reporter activity of pNGL cell lines stably expressing pC, pChuk, pIkbkb, pIkbke, and pTbk1, (n = 3 independent experiments). d Bioluminescent detection of NF-κΒ reporter activity in LLC, MC38, and AE17 cells (KrasMUT) stably expressing pNGL and shC, shChuk, or shIkbkb (n = 3 independent experiments) during 4-h incubation with 1 nM IL-1β. Note IL-1β-induced NF-κB activity of shC and shIkbkb cells that is silenced in shChuk cells. e Immunoblots of cytoplasmic and nuclear protein extracts of MC38 cells stably expressing pNGL and shC, shChuk, or shIkbkb after 4-h treatment with PBS, TNF, and IL-1β for various NF-κB pathway members and β-actin (n = 3). Data are presented as mean ± s.d. of n = 3 independent experiments. P, probability of no difference between cell lines by two-way ANOVA. ns, single, double, and triple asterisks (*, **, and ***): P > 0.05, P < 0.05, P < 0.01, and P < 0.001, respectively, for comparison of color-coded sh or p with control sh or p within each cell line by Bonferroni post-tests
