Fig 2: Cardamonin blocks both ß-catenin and NF-?B pathways and abrogates TLR3-activation-induced CSCs in vitro. (a) Molecular structure of cardamonin (2,4-dihydroxy-6-methoxychalcone) (b) Representative images of SUM190 cells treated with 1 µg/ml of poly(I:C) in the presence or absence of cardamonin (10 µM) for 4 days. Cardamonin abrogates the formation of non-adherent spherical clusters induced by poly(I:C) stimulation. Scale bars, 100 µm. (c) Western blot analysis of nuclear translocation of NF-?B p65 and active ß-catenin. SUM190 cells were pretreated with 10 µM of cardamonin for 4 h, followed by stimulation with poly(I:C) (1 µg/ml) for 4 days in the presence of cardamonin. a-tubulin and Lamin A/C: internal loading controls for cytoplasmic (Cyto) and nuclear (Nu) proteins, respectively. (d) Flow-cytometry analysis of CD44high/CD24-/low sub-population in four different breast cancer cell lines treated as described in (c). Data represent the average±S.D., n=3; *P<0.05. (e) Western blot analysis of c-Myc, CD44, and Oct4 proteins for the indicated groups as shown in (c). a-tubulin and Lamin A/C were included as internal loading controls for cytoplasmic (Cyto) and nuclear (Nu) extracts, respectively. (f) Soft-agar colony formation assay to evaluate tumorigenic potential. SUM190 cells were treated as described in (c), and seeded in soft agar in 12-well plates (5 × 103 cells/well) for 17 days. Cellular aggregates with a diameter of >100 µm were evaluated as colonies after staining with MTT for live cells. Data were expressed as fold increase over the control value (which was arbitrarily set to 1). Scale bar, 100 µm. Data represent the average±S.D., n=4; *P<0.05

Fig 3: Interferon-? signaling synergizes with LRRK2 in human neurons and microglia.IFN-? response is conserved in DA neurons, and IFN-? induces LRRK2 expression in DA neurons and microglia (a). The PD-associated mutation LRRK2 G2019S sensitizes DA neurons to IFN-? by decreasing AKT phosphorylation (b) and suppressing NFAT nuclear shuttling (c) that, in turn, leads to defects in neurite outgrowth (d). LRRK2-dependent defects of NFAT translocation are linked to defects in calcium buffering capacity (e) and possibly to an increased complexity of the microtubule network (f). Defects in NFAT shuttling are also observed in LRRK2 G2019S microglia (g), which display a more activated phenotype upon stimulation. LRRK2 G2019S modulates microglia activation by interfering with the metabolic switch toward glycolysis that normally occurs upon mononuclear phagocyte activation (h) and impairing NF-?B p65 nuclear translocation upon stimulation with IFN-? or LPS (i).

Fig 4: ECRG4 inhibits the NF-?B pathway via the upregulation of OGN in BCa cells. (A) NF-?B transcriptional activity was detected using an NF-?B luciferase reporter gene system. (B) Western blotting detected the protein levels of ECRG4, OGN, p65, and p-p65 (S536) in BCa cells after transfection with ECRG4-overexpressing or silencing plasmids. (C,D) Quantification of the protein levels of ECRG4, OGN, p65, and p-p65 (S536) in panel (B). (E) The NF-?B luciferase reporter gene system was used to detect the NF-?B transcriptional activity of BCa cells after transfection with indicated plasmids. (F) Western blotting detected the protein levels of p65 and p-p65 (S536) in BCa cells after transfection with the indicated plasmids. (G) Quantification of the protein levels of p65 and p-p65 (S536) in panel (F). (H) The proposed mechanism of ECRG4 inhibition of the carcinogenesis of BCa cells via NFIC/OGN/NF-?B signaling. (A–G) ANOVA followed by Bonferroni’s post hoc test. All data were replicated three times. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig 5: β-Catenin and NF-κB co-mediate TLR3 activation-induced CSC phenotypes. (a) qPCR analysis of target genes of β-catenin pathway (Axin2 and CylinD1) in SUM190 after treatment with 1 μg/ml of poly(I:C) for 4 days. Data represent the average±S.D., n=5; *P<0.05. (b) Western blot analysis of nuclear translocation of active β-catenin in SUM190 cells after treatment with poly(I:C) (1 μg/ml ) for 4 days. α-tubulin and Lamin A/C: internal loading controls for cytoplasmic (Cyto) and nuclear (Nu) proteins. (c) qPCR analysis of the indicated stem markers. SUM190 cells were pretreated for 4 h with Bay 11-7821 (5 μM, an NF-κB inhibitor) and BC21 (7.5 μM, a β-catenin/TCF4 inhibitor) either alone or in combination, followed by stimulation with poly(I:C) for 4 days in the presence or absence of the above inhibitors. Data represent the average±S.D., n=3; *P<0.05. (d) Inhibition of both NF-κB and β-catenin pathways completely blocks poly(I:C)-induced expression of CD44 and c-Myc (western blot). SUM190 cells were treated in the same way as those described in (c). (e) SUM190 cells were transfected with siRNA oligos against NF-κB p65 (p65), β-catenin or non-targeting oligos for 24 h, and then treated with 1 μg/ml of poly(I:C) for 3 days. Immunoblotting shows an almost complete knockdown of NF-κB and β-catenin expression after co-transfection of NF-κB p65 and β-catenin siRNA. (f) Knockdown of both NF-κB (p65) and β-catenin abrogates an increase in CD44high/CD24−/low cell population following poly(I:C) treatment. Data represent the average±S.D., n=3; *P<0.05