Fig 1: MARCH5-dependent NLRP3 ubiquitination allows the binding of NEK7 to NLRP3 ARepresentative confocal images of oligomerized NLRP3. ASC knock-downed immortalized BMDMs expressing GFP-tagged NLRP3 WT were transfected using indicated siRNAs, and treated with 200 ng/ml LPS for 4 h followed by 15 µM nigericin for 30 min. White arrows indicate oligomerized NLRP3. The percentage of cells with oligomerized NLRP3 was quantified. At least 100 cells were analyzed. Bar, 10 µm. Values are the mean ± SD. *P < 0.05 (two-tailed Student's t-test).B March5 fl/fl and March5 fl/fl;Lyz-Cre BMDMs were stimulated for 4 h with LPS followed by 5 mM ATP or 15 µM nigericin for the indicated durations. SDD–AGE and SDS–PAGE were performed to separate cell lysates, followed by immunoblotting using indicated antibodies.CTHP-1 cells transfected with siControl or siMARCH5 were stimulated with 200 ng/ml LPS for 4 h followed by 5 mM ATP for 30 min. Cell lysates were subjected to immunoprecipitation by using NLRP3 antibody. Western blotting was performed using the indicated antibodies.DImmortalized BMDMs were transfected with siControl or siMARCH5 and stimulated with LPS and nigericin. Blue native PAGE was used first to separate the cell lysates, and then SDS–PAGE was used to separate them further. Western blotting was performed using the indicated antibodies. Each yellow circle represents the oligomerized form of indicated protein.EHEK293T cells were transfected with FLAG-tagged NLRP3 WT or indicated mutants and stimulated with LPS and nigericin. Cell lysates were immunoprecipitated with FLAG-M2 beads and detected with the indicated antibodies.FHEK293T cells were transfected with FLAG-tagged NLRP3 WT or indicated mutants. Cells were stimulated with LPS and nigericin. Cell lysates were separated by blue-native PAGE (first dimension) and SDS–PAGE (second dimension). Representative data from independent experiments that were repeated at least three times are shown. Each yellow circle represents the oligomerized form of indicated protein.GRepresentative confocal image showing NLRP3-NEK7 colocalization. siControl- and siMarch5- transfected ASC knock-downed iBMDMs expressing GFP-tagged NLRP3 WT cells were stimulated with 200 ng/ml LPS for 4 h followed by 15 µM nigericin for 30 min. The percentage of cells with NLRP3-NEK7 colocalization was quantified. At least 100 cells were analyzed. Values are shown as the mean ± SD. ***P < 0.001 (two-tailed student's t-test). See also Appendix Figure S1. Bar, 10 µm. Source data are available online for this figure.
Fig 2: MARCH5-mediated ubiquitination of NLRP3 is essential for inflammasome activation AHEK293T cells were transfected with NLRP3 WT or indicated Lys mutants and ASC. Cells were stimulated with 200 ng/ml LPS for 4 h followed by 15 µM nigericin for 30 min. The percentage of cells with an ASC speck was quantified after Confocal microscopy. At least 100 cells were analyzed. Values are the mean ± SD. Values, **P < 0.01, ***P < 0.001 (two-tailed Student's t-test). Bar, 10 µm.BHEK293T cells were transfected with ASC and NLRP3 (WT or individual NLRP3 mutants). Cells were treated for 4 h with 200 ng/ml LPS and 30 min with 15 µM nigericin. Harvested cell lysates were incubated with 2 mM DSS for cross-linking. Triton X-100 insoluble pellets and soluble fraction lysates were detected by immunoblotting with the indicated antibodies.CNLRP3 inflammasome-reconstituted HEK293T cells with NLRP3 WT or indicated mutants were stimulated for 4 h with 200 ng/ml LPS and 30 min with 15 µM nigericin. Harvested cells were detected by immunoblotting with the indicated antibodies.DCulture supernatants were obtained from (C) and subjected to ELISA to quantify secreted IL-1ß. Representative data are shown from independent experiments that were repeated at least three times. Values are the mean ± SD. *P < 0.05, ***P < 0.001 (two-tailed Student's t-test)ESchematic diagram of NLRP3 inflammasome activation by MARCH5. Upon activation by a priming signal, NLRP3 associates with MAVS on mitochondria ?. MARCH5 recognizes and ubiquitinates two lysine sites, K324 and K430, on the NLRP3 NACHT domain via K27-linked polyubiquitination ?. NLRP3 ubiquitination by MARCH5 promotes the recruitment of NEK7 and enables self-oligomerization ?. As a result, ASC and pro-caspase-1 are recruited to oligomerized NLRP3 ? for NLRP3 inflammasome assembly ? and trigger the activation of caspase-1 and pro-cytokine cleavage ?. Source data are available online for this figure.
Supplier Page from Sino Biological, Inc. for Human NEK7 Gene ORF cDNA clone expression plasmid, C-HA tag