Fig 1: The C-terminal of CYPJ interacts with TAB2 and TAB3. a Effects of CYPJ on NF-?B reporter activation induced by individually transfected components of the NF-?B signal pathway (N = 3). b, c Reciprocal co-IP assays determine the interaction between HA-CYPJ and Flag-TAB2 or Flag-TAB3 in 293T cells. d Endogenous co-IP assays evaluate the interaction between CYPJ and TAB2/TAB3 with or without TNF treatment for different time points in 293T cells. e Immunofluorescence microscopy observing the colocalization of pEGFP-CYPJ and Flag-TAB2 in HeLa cells. Scale bars, 20 µm. f Multiple sequence alignments of CYPJ proteins from different species. The three fragments of CYPJ are marked. g GST pull-down assays were performed using E.coli-expressed GST and GST-CYPJ fragments and lysates of 293T cells transfected with Flag-TAB2 or Flag-TAB3. h Co-IP assays detecting the interaction between Flag-TAB2/3 with HA-tagged FL or fragment-3 deleted CYPJ, # indicates a nonspecific band. Error bars indicate S.D.; n.s. no significance, * p < 0.05, ** p < 0.01 (two-tailed Student’s t-test)
Fig 2: CYPJ negatively regulates the NF-?B signal pathway. a CYPJ inhibits TNF-induced activation of the NF-?B reporter in 293T cells (N = 3). b CYPJ inhibits TNF-induced transcription of TNF and IL-8 in 293T cells (N = 3). c CRISPR-Cas9-mediated CYPJ deficiency (inset) enhances TNF-induced activation of the NF-?B reporter in 293T cells (N = 3). d CYPJ knockout enhances TNF-induced transcription of TNF and IL-8 in 293T cells (N = 3). e 293T cells were transfected with a control or HA-CYPJ plasmid and were treated with TNF for different times. Phosphorylation of the indicated proteins was detected. f WT and CYPJ-deficient 293T cells were treated with or without TNF, and phosphorylation of the indicated proteins was detected. g HeLa cells were transfected with pEGFP-CYPJ (green) and treated with or without TNF (15 ng ml-1) for 15 min. The cells were fixed, permeabilized, and stained for p65 (red) and 4'6-diamidino-2-phenylindole (DAPI) (blue), followed by observation by laser confocal fluorescence microscopy. Scale bars, 20 µm. The white arrows indicate GFP-CYPJ transfected cells. h 293T cells were transfected with or without HA-CYPJ and stimulated by TNF (15 ng ml-1) for 15 min. The cytoplasmic and nuclear proteins were isolated and detected with the indicated antibodies. i–k Protein levels of CYPJ and CYPA were detected in TNF-treated HeLa cells (i), LPS-treated primary mBMM cells (j), and VSV-infected A549 cells (k). Error bars indicate the standard deviation (S.D.); n.s. no significance, * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed Student’s t-test)
Fig 3: Cypj protects mice from lethal acute inflammation and DSS-colitis. a, b Survival curve (a N = 6 mice per group, Log-rank Test) and ELISA of serum Tnf level (b N = 5 mice per group, two-tailed Student’s t-test) in Cypj-deficient and WT mice after intraperitoneal injection of LPS (10 mg per kg body weight). c Histopathology of lungs from Cypj-deficient and control mice after intraperitoneal injection of LPS (10 mg kg-1). Scale bars, 100 µm. d Survival curve (N = 7 mice per group, Log-rank test) of WT and Cypj KO mice after intraperitoneal injection of HKLM (2 × 1012 CFU per mice). e Weight loss during 3% DSS-induced colitis was expressed as the percentage of change from day 0 (N = 5 mice per group, two-tailed Student’s t-test). f Colon lengths of DSS-treated mice (N = 5 mice per group, two-tailed Student’s t-test) were assessed at the time of necropsy, with representative macroscopic images and statistics. g Representative micrographs of colon hematoxylin and eosin (H&E) staining from WT and KO mice 7 days after DSS-colitis. Scale bars, 100 µm. h Proposed working model of CYPJ dependent negative feedback regulation of the NF-?B signal pathway. Error bars indicate S.D.; * p < 0.05, *** p < 0.001
Fig 4: The C-terminus is required for CYPJ to inhibit NF-?B signaling. a, b Effects of CYPJ and CYPJ-?3 on the activity of NF-?B reporters induced by TNF (a N = 3) or TAB2/3 (b N = 3). c, d Effects of CYPJ and CYPJ-?3 on TNF-induced transcription of TNF and IL-8 in the CYPJ-knockout 293T cell lines KO-1 (c N = 3) and KO-2 (d N = 3). e, f Effects of CYPJ and CYPJ-?3 overexpression on TNF-induced phosphorylation of indicated proteins in two CYPJ-knockout cell lines cell lines, KO-1 (e) and KO-2 (f). g, h Fragment-3 is required for CYPJ to disrupt the binding of K63-Ub chain to TAB2 (g) and TAB3 (h). Plasmids were transfected into 293T cells as indicated and the binding of polyubiquitin chain to TAB2/3 was detected with an anti-HA antibody after IP with an anti-Flag antibody. Error bars indicate S.D.; * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed Student’s t-test)
Fig 5: Cypj deficiency activates the NF-?B signaling in primary mBMM cells. a The strategy of CRISPR-Cas9 mediated genomic edition. A gene specific guide RNA (sgRNA) was used to achieve the targeting. b, c Percentage of mBMMs (b) and mBMDC (c) were analyzed by their specific lineage markers between WT and KO mice. d–i WT and Cypj-deficient primary mouse BMM cells were treated with or without LPS (100 ng ml-1) (d, e), Tnf (20 ng ml-1) (f, g), or HKLM (107 CFU ml-1) (h, i) for different times; Cypj abundance and the phosphorylation of the indicated proteins (d, f, h), as well as transcription of Tnf and Il-6 (e, g, i; N = 3) were detected. Error bars indicate S.D.; * p < 0.05, ** p < 0.01 (two-tailed Student’s t-test)
Supplier Page from Abcam for Anti-PPIL3 antibody