Journal: bioRxiv

Article Title: Cullin1 represses systematic inflammasome activation by binding and catalyzing NLRP3 ubiquitination

doi: 10.1101/289637

Figure Lengend Snippet: A HEK293T cells were transfected or co-transfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1β, pcDNA3.1(+)-pro-caspase-1, and pcDNA3.1(+)-ASC, as indicated. B HEK293T cells were co-transfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1β, pcDNA3.1(+)-pro-caspase-1, and pcDNA3.1(+)-ASC, along with pcDNA3.1(+)-3Flag-ATP1β3, pcDNA3.1(+)-3Flag-PGM1, or pcDNA3.1(+)-3Flag-Cullin1, as indicated. Secreted IL-1β in the supernatants were analyzed by ELISA. C Daigrams of Cullin1 and Cullin1ΔC (upper). HEK293T cells were co-transfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1β, pcDNA3.1(+)-pro-caspase-1, and pcDNA3.1(+)-ASC along with pcDNA3.1(+)-3XFlag-Cullin1 or pcDNA3.1(+)-3XFlag-Cullin1ΔC, as indicated. Secreted IL-1β in the supernatants were analyzed by ELISA. D HEK293T cells were co-transfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1β, pcDNA3.1(+)-pro-caspase-1, and pcDNA3.1(+)-ASC along with pcDNA3.1(+)-3XFlag-Cullin1 or pcDNA3.1(+)-3XFlag-Cullin1ΔC. E HEK293T cells were co-transfected with pCAGGS-HA-NLRP3 and pcDNA3.1(+)-3XFlag-Cullin1, pcDNA3.1(+)-3XFlag-Cullin2, or pcDNA3.1(+)-3XFlag-Cullin3, respectively. The cell lysates were immunoprecipitated with anti-HA antibody and then immunoblotted with indicated antibodies. F HEK293T cells were co-transfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-ASC, pcDNA3.1(+)-pro-caspase-1, and pcDNA3.1(+)-pro-IL-1β along with pcDNA3.1(+)-3XFlag-Cullin1, pcDNA3.1(+)-3XFlag-Cullin2, or pcDNA3.1(+)-3XFlag-Cullin3, respectively. G HEK293T cells were transduced with lentiviruses stably expressing shRNA (sh-NC) and shRNA against Cullin1 (sh-Cullin1#1 and sh-Cullin1#2) and selected with puromycin for 2 weeks. Cullin1 and GAPDH mRNAs were determined by qRT-PCR (upper) and Cullin1 and GAPDH proteins were detected Western blot analyses (low). H HEK293T cells stably expressing sh-NC or sh-Cullin1#2 were co-transfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-pro-IL-1β, pcDNA3.1(+)-pro-caspase-1, and pcDNA3.1(+)-ASC, along with pcDNA3.1(+)-3XFlag-Cullin1. Data information: In ( A , D , F , and H ), secreted IL-1β in the supernatants were analyzed by ELISA (upper). Matured IL-1β (p17) and matured Casp-1 (p20) in the cell lysates were determined by immunoblot analyses with indicated antibodies (low). Data information: In ( A–D , G–H ), data shown are means ± SEM, *P<0.05, **P<0.01, ***P<0.0001.

Article Snippet: Human IL-1β/IL-1F2 Quantikine ELISA Kit (Cat# DLB50) was purchased from R&D Systems (Minnesota, USA).

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Transduction, Stable Transfection, Expressing, shRNA, Quantitative RT-PCR, Western Blot

Journal: bioRxiv

Article Title: Cullin1 represses systematic inflammasome activation by binding and catalyzing NLRP3 ubiquitination

doi: 10.1101/289637

Figure Lengend Snippet: A PMA-differentiated THP-1 macrophages were infected with H3N2 for 24 h or treated with LPS and ATP for 30 min. The cell lysates were immunoblotted with indicated antibodies. B CUL1, Cyclin E1, and GAPDH proteins were examined by Western blot analysis in THP-1 cells stably expressing CUL1. C Macrophages stably expressing CUL1 were treated with ATP for 30 min. D Macrophages stably expressing CUL1ΔC were treated with ATP or infected with H3N2. Secreted IL-1β in the supernatants were analyzed by ELISA. E Macrophages stably expressing sh-Cullin1#2 were treated with LPS for 6 h, LPS for 6 h and ATP for 30 min, or LPS for 6 h and Nigericin for 1 h, respectively. Matured IL-1β (p17) in the cell lysates was determined by immunoblot analysis with indicated antibodies. F Macrophages stably expressing sh-Cullin1#2 were treated with ATP for 30 min. G Macrophages stably expressing sh-Cullin1#2 were treated with Nigericin for 1 h. H Macrophages stably expressing sh-Cullin1#2 were treated with Alum for 6 h I Macrophages stably expressing sh-Cullin1#2 were infected with IAV H3N2 for 24 h J Macrophages stably expressing sh-Cullin1#2 were treated with Poly(dA:dT)/LyoVec TM for 12 h. Data information: In ( C , F–J ), secreted IL-1β in the supernatants were analyzed by ELISA (upper). Matured IL-1β (p17) and matured Casp-1 (p20) in the cell lysates were determined by immunoblot analysis with indicated antibodies (low). Data information: In ( C , D , F–J ), data shown are means ± SEM, *P<0.05, **P<0.01, ***P<0.0001.

Article Snippet: Human IL-1β/IL-1F2 Quantikine ELISA Kit (Cat# DLB50) was purchased from R&D Systems (Minnesota, USA).

Techniques: Infection, Western Blot, Stable Transfection, Expressing, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Cullin1 represses systematic inflammasome activation by binding and catalyzing NLRP3 ubiquitination

doi: 10.1101/289637

Figure Lengend Snippet: A HEK293T cells were transfected with pcDNA3.1(+)-3XFlag-Cullin1 and pCAGGS-HA-NLRP3, pCAGGS-HA-PYD, pCAGGS-HA-NBD, pCAGGS-HA-LLR, respectively. B HEK293T cells were transfected with pCAGGS-HA-Cullin1 and pcDNA3.1(+)-3XFlag-NLRP3, pcDNA3.1(+)-3XFlag-ASC, or pcDNA3.1(+)-3XFlag-pro-caspase-1, respectively. C HEK293T cells were transfected with pcDNA3.1(+)-NLRP3 and pcDNA3.1(+)-ASC along with pcDNA3.1(+)-3XFlag-Cullin1. D HEK293T cells were transfected with pCAGGS-HA-NLRP3 and pCAGGS-HA-ASC along with pcDNA3.1(+)-3XFlag-Cullin1 at different concentrations. E HEK293T cells were co-transfected with pcDNA3.1(+)-NLRP3 and/or pcDNA3.1(+)-ASC along with pcDNA3.1(+)-3XFlag-Cullin1, as indicated. F HEK293T cells were co-transfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-ASC, pcDNA3.1(+)-3XFlag-Cullin1 and then treated with 5 mM ATP for 30 min and 10 μM Nigericin for 1 h. G PMA-differentiated THP-1 macrophages were stimulated with ATP or Nigericin, and infected with H3N2. H PMA-differentiated THP-1 macrophages were stimulated with ATP or Nigericin. The cells were immunostained with anti-NLRP3 and anti-Cullin1 antibodies. The sub-cellular localizations of endogenous NLRP3 (green), endogenous Cullin1 (Red), and nucleus marker DAPI (blue) were analyzed under confocal microscopy. I PMA-differentiated THP-1 macrophages were stimulated with ATP or Nigericin. The cells were immunostained with anti-NLRP3 and anti-Cullin1 antibodies. The supernatants were analyzed by ELISA for IL-1β secretion. Data shown are means ± SEM, *p<0.05, **p<0.01, ***p<0.0001. Data information: In ( A–G ), the cell lysates were immunoprecipitated with anti-HA antiboody (A), anti-Flag antibody (B, C), anti-NLRP3 antibody (D, F), or anti-Cullin1 antibody (E, G) and then immunoblotted with indicated antibodies.

Article Snippet: Human IL-1β/IL-1F2 Quantikine ELISA Kit (Cat# DLB50) was purchased from R&D Systems (Minnesota, USA).

Techniques: Transfection, Infection, Marker, Confocal Microscopy, Enzyme-linked Immunosorbent Assay, Immunoprecipitation

Journal: bioRxiv

Article Title: Cullin1 represses systematic inflammasome activation by binding and catalyzing NLRP3 ubiquitination

doi: 10.1101/289637

Figure Lengend Snippet: A HEK293T cells were transfected with pcDNA3.1(+)-NLRP3 and pCAGGS-HA-Ub at different concentrations. B HEK293T cells were transfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-HA-Ub, and/or pcDNA3.1(+)-3Flag-Cullin1, respectively. C HEK293T cells were transfected with pcDNA3.1(+)-NLRP3, pcDNA3.1(+)-HA-Ub, pcDNA3.1(+)-3Flag-Cullin1 or pcDNA3.1(+)-3Flag-ROC1, as indicated. D Daigrams of full-length NLRP3 and its point mutants. The potential ubiquitination sites in NLRP3 among the Lys residues were predicated by using UbPred software. One Lys (Lys689) is high confidence and six Lys (Lys93, Lys192, Lys194, Lys324, Lys430, and Lys696) are medium confidence. Seven mutations of CUL1 (K93R, K192R, K194R, K324R, K430R, K689R, and K696R), in which the K residues were replaced by R residues. E HEK293T cells were co-transfected with pcDNA3.1(+)-HA-Ub and pcDNA3.1(+)-Cullin1, along with pcDNA3.1(+)-3xFlag-NLRP3 or its mutants, respectively. F HEK293T cells were co-transfected with pcDNA3.1(+)-NLRP3 and/or pcDNA3.1(+)-HA-Ub, pcDNA3.1(+)-3Flag-Cullin1, pcDNA3.1(+)-HA-K48 or pcDNA3.1(+)-HA-K63, as indicated. G PMA-differentiated THP-1 macrophages stably expressing sh-NC and sh-Cullin1#2 were infected with IAV H3N2. H HEK293T cells were transfected with pcDNA3.1(+)-3XFlag-NLRP3, pcDNA3.1(+)-3XFlag-ASC, pcDNA3.1(+)-3XFlag-pro-caspase-1, pcDNA3.1(+)-3XFlag-pro-IL-1β, or pcDNA3.1(+)-3xFlag-Cullin1, as indicated. I HEK293T cells were transfected with pCAGGS-HA-NLRP3 and/or pcDNA3.1(+)-3XFlag-Cullin1, or pcDNA3.1(+)-3XFlag-ROC1. Data information: In ( A–C ), the lysates were immunoprecipitated with anti-NLRP3 antibody and immunoblotted with indicated antibodies. In ( E–I ), the lysates were immunoprecipitated with anti-Flag antibody (E) or anti-NLRP3 antibody (F, G) and then immunoblotted with indicated antibodies. The lysates were immunoblotted with the indicated antibodies (H, I).

Article Snippet: Human IL-1β/IL-1F2 Quantikine ELISA Kit (Cat# DLB50) was purchased from R&D Systems (Minnesota, USA).

Techniques: Transfection, Software, Stable Transfection, Expressing, Infection, Immunoprecipitation

Journal: bioRxiv

Article Title: Cullin1 represses systematic inflammasome activation by binding and catalyzing NLRP3 ubiquitination

doi: 10.1101/289637

Figure Lengend Snippet: A L929 cells were transfected with siR-NC (25 nM), siR-Cullin1#1 (25 nM), siR-Cullin1#2 (25 nM), or siR-Cullin1#3 (25 nM). Cullin1 and GAPDH mRNAs were determined by qRT-PCR (left) and CUL1 and GAPDH proteins were detected Western blot analyses (right). B C57BL/6 mice were injected with siR-NC (5 nM), siR-Cullin1#1 (5 nM), or siR-Cullin1#1 (10 nM) for 60 h, and the peritonitis of treated mice were then injected with Alum for 12 h. The endogenous mCUL1, mNLRP3, and mGAPDH proteins in the mice peritoneal exudates cells (PECs) were detected by Western blot analyses (left). Secreted IL-1β in the mice sera were analyzed by ELISA (right). C C57BL/6 mice (six mice per group) were injected with siR-NC or siR-Cullin1#1 for 60 h, and the peritonitis of treated mice were then injected with Alum. Secreted IL-1β in mice peritoneal lavage fluid were analyzed by ELISA. D C57BL/6 mice (six mice per group) were injected with siR-NC or siR-Cullin1#1 for 60 h, and the peritonitis of treated mice were then injected with Alum. Secreted IL-6 in mice peritoneal lavage fluid was analyzed by ELISA. E C57BL/6 mice (six mice per group) were injected with siR-NC or siR-Cullin1#1 for 60 h, and the peritonitis of treated mice were then injected with Alum. The total numbers of PECs in peritoneal cavity were stimulated. F C57BL/6 mice (six mice per group) were injected with siR-NC or siR-Cullin1#1 for 60 h, and the peritonitis of treated mice were then injected with Alum. The total numbers of Neutrophils (F) in peritoneal cavity were stimulated. G C57BL/6 mice (six mice per group) were injected with siR-NC or siR-Cullin1#1 for 60 h, and the peritonitis of treated mice were then injected with Alum. Histopathological changes in the mice spleen tissues were examined by H&E staining. Data information: In ( A–F ), data shown are means ± SEM, *P<0.05, **P<0.01, ***P<0.0001.

Article Snippet: Human IL-1β/IL-1F2 Quantikine ELISA Kit (Cat# DLB50) was purchased from R&D Systems (Minnesota, USA).

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Injection, Enzyme-linked Immunosorbent Assay, Staining

Journal: bioRxiv

Article Title: Cullin1 represses systematic inflammasome activation by binding and catalyzing NLRP3 ubiquitination

doi: 10.1101/289637

Figure Lengend Snippet: A In resting cells and under normal physiological conditions, CUL1 interacts with NLRP3 to disrupt the NLRP3 inflammasome assembly, and catalyzes NLRP3 ubiquitination to repress the NLRP3 inflammasome activation. B In response to and inflammasome stimuli and pathogen infection, CUL1 disassociates from NLRP3 to release the repression of NLRP3 inflammasome assembly and activation. ASC binds NLRP3 to form ASC oligomers, which provides a platform for activation of pro-Casp-1 that regulates the maturation and secretion of IL-1β and IL-18, which in turn initiate multiple signaling pathways and drive inflammatory responses.

Article Snippet: Human IL-1β/IL-1F2 Quantikine ELISA Kit (Cat# DLB50) was purchased from R&D Systems (Minnesota, USA).

Techniques: Activation Assay, Infection