Fig 2: CNTN1 negatively regulates the type I IFN signaling pathway.(A-B) CNTN1 inhibits IFN-related promoter activities. Luciferase reporter plasmids (IFN-ß-Luc or ISRE-Luc) and the pRL-TK plasmid were co-transfected into HEK293 cells, along with Flag-CNTN1 or Vec (A) or CNTN1 siRNA or scrambled siRNA (NC) (B). At twenty-four hours after transfection, the cells were left uninfected or were infected with SeV for 12 h before reporter assays. (C) Luciferase reporter plasmids (IFN-ß-Luc or ISRE-Luc) and the pRL-TK plasmid were co-transfected into HEK293 cells, along with different concentrations of CNTN1 expression plasmid. At twenty-four hours after transfection, cells were left uninfected or were infected with SeV for 12 h before reporter assays. (D) Luciferase reporter plasmids (IRF1-Luc) and the pRL-TK plasmid were co-transfected into HEK293 cells, along with Flag-CNTN1 or Vec or CNTN1 siRNA or NC. At twenty-four hours after transfection, the cells were left untreated or were treated with IFN-ß for 12 h before reporter assays. (E-F) A549 cells were transfected with Flag-CNTN1 or Vec (E) or CNTN1 siRNA or NC (F), and subsequently left uninfected or infected with SeV for 12 h before qPCR analysis. (G) CNTN1 inhibits virus-induced phosphorylation of TBK1 and IRF3. A459 cells were transfected with GFP-CNTN1 plasmid or Vec. At twenty-four hours after transfection, the cells were infected with SeV for the indicated times. Then, immunoblotting analysis were performed with the indicated antibodies. (H) HEK293 cells were transfected with CNTN1 or Vec, and induced by RIG-IN transfection. The concentration of IFN-ß in the supernatants was detected by an ELISA. (I) IFNAR1+/+ or IFNAR1-/- A549 cells were transfected with the CNTN1 expression plasmid or Vec. Twenty-four hours later, the cells were infected with H5N6 virus at an MOI of 0.01 or 3. At the indicated times post-infection, the supernatant containing viral particles was assessed in the TCID50 assay. The significant difference between IFNAR1+/+-Vec and IFNAR1+/+-CNTN1 is labeled in red; the significant difference between IFNAR1+/+-Vec and IFNAR1-/--Vec is labeled in blue; the significant difference between IFNAR1-/--Vec and IFNAR1-/--CNTN1 is labeled in green. The data shown represent three independent experiments; bars represent the mean ± SD of the three independent experiments (n = 3). [P< 0.05 (*), P < 0.01 (**), P < 0.001 (***), P < 0.0001 (****); ‘ns’ indicates no significant difference].

Fig 3: BAI reduced LPS-induced damage via TLR4/NF-?B signaling. The protein expressions of TLR4/NF-?B signaling were established by (A-D) western blotting. (A) Blots for LPS stimulation demonstrated that (B) TLR4, p-TAK1, p-TBK1 and p-p65 were upregulated by LPS, then downregulated by BAI treatment. (C) MRT67307 treatment and (D) inhibited the effect of BAI on p-TBK1. (E) The immunofluorescent staining of p65 (magnification, ×200) revealed that (F) there was more cell nuclear translocation with LPS stimulation and less with BAI treatment. The data are expressed as the mean ± standard deviation. *P<0.05 vs. control (0 µM BAI/LPS); #P<0.05 vs. LPS group (10 µM LPS only); **P<0.05 vs. LPS+BAI (10 µM LPS, 6.25 µM BAI). BAI, baicalein; LPS, lipopolysaccharide; HUVECs, human umbilical vein endothelial cells; TLR4, Toll-like receptor 4; p, phosphorylated; TAK1, transforming growth factor ß-activated kinase 1; TBK1, tumor necrosis factor receptor-associated family member associated nuclear factor-?B activator-binding kinase 1.

Fig 4: ERK1/2 activations are abrogated in IKK? deficient tumor cells from various tissues. (A) ERK1/2 and MEK1 constitutive phosphorylations are diminished in IKBKE deficient IECs from Apc+/min mice but only in tumor samples. (B) Transformed MEF cells deficient of only IKBKE (but not in IKKa or IKKß) also show abrogated ERK1/2 phosphorylation (where different genotypes of MEF cells were stimulated 0-30 minutes, at indicated time points, with 10 ng/ml LPS). (C-E) Tumor cell lines from various epithelial neoplasia also show diminished ERK1/2 phosphorylation upon loss of IKBKE via siRNA knockdown after stimulated with respective ligands (where DLD-1 and HCT116 cells were stimulated 0-30 minutes, at indicated time points, with 10 ng/ml LPS and HeLa cells were treated 0-2 hours, at indicated time points, with 10 µg/ml Poly I:C). (F-H) On the contrary, ERK1/2 activations in normal cells were not affected from IKK? deficiency. (F) RAW264.7 cells and (G) BMDMs were treated with 10 ng/ml LPS, as indicated in each panel while (H) Ikbke wt or ko mice were injected with 0.1 mg/kg LPS 0-8 h, at indicated time points, for intestinal epithelial cell stimulation. ERK1/2 phosphorylation was measured by WB using a phospho-specific antibody. (I) Impact of TBK1 depletion on ERK1/2 phosphorylations were evaluated in CRC cell lines. To do this, ERK1/2 phosphorylation was examined in lentiviral shRNA (shCTRL vs shTBK1 (1-4)) transfected SW480 and HCT116 cells via WB.

Fig 5: Effects of IFI16, H2B, BRCA1, cGAS, STING and ASC knockdown on IFN-ß during KSHV and HSV-1 infection.(A) WCL from HMVEC-d cells electroporated with siC, siH2B, siIFI16, siSTING or siASC for 48 h followed by infection for 4 h with or without KSHV (KS) were western blotted with anti-IFI16, ASC, H2B and STING antibodies. (B) Phosphorylation of TBK1 and IRF3 in H2B, IFI16, BRCA1, cGAS, STING and ASC knockdown cells infected with KSHV. HMVEC-d cells electroporated with different siRNA (panel A and Fig 6F) for 48 h were followed by with or without KSHV infection (4 h), and immunoblotted using anti-pTBK1, tTBK1 (total), pIRF3 and tIRF3 antibodies. (C and D) Effect of IFI16, H2B, BRCA1, cGAS, STING and ASC knockdown on IFN-ß and IL-1ß secretion during KSHV de novo infection. (C) Cell culture supernatants from HMVEC-d cells electroporated with siC, siIFI16, siBRCA1, sicGAS, siSTING, siH2B or siASC for 48 h followed by infection (4 h) with or without KSHV (Fig 7A and Fig 6F), were collected and subjected to IFN-ß ELISA. Results presented are means ± SD (* p<0.05, ** p<0.01 from siC vs. siIFI16, siBRCA1, sicGAS, siSTING, and siH2B with KSHV infection; NS: not significant). (D) The same supernatants from panel C were used to detect IL-1ß secretion by ELISA. Results presented are means ± SD (* p<0.05, from siC vs. siIFI16, and siASC with KSHV infection; NS: not significant). (E) Effect of IFI16, H2B, BRCA1, cGAS, STING and ASC knockdown on IFN-ß secretion during HSV-1 infection. Cell culture supernatants from HFF cells electroporated with siRNA (S6F Fig) for 48 h followed by with or without HSV-1 infection (4 h) were collected and IFN-ß ELISA was performed. Results presented are means ± SD (*** p<0.001 from siC vs. siIFI16, siBRCA1, sicGAS, siSTING, or siH2B with HSV-1 infection; NS: not significant).