SLC25A3 negatively regulates NLRP3 inflammasome activation by restricting the function of NLRP3

The NACHT, leucine-rich repeat, and pyrin domains-containing protein 3 (collectively known as NLRP3) inflammasome activation plays a critical role in innate immune and pathogenic microorganism infections. However, excessive activation of NLRP3 inflammasome will lead to cellular inflammation and tissue damage, and naturally it must be precisely controlled in the host. Here, we discovered that solute carrier family 25 member 3 (SLC25A3), a mitochondrial phosphate carrier protein, plays an important role in negatively regulating NLRP3 inflammasome activation. We found that SLC25A3 could interact with NLRP3, overexpression of SLC25A3 and knockdown of SLC25A3 could regulate NLRP3 inflammasome activation, and the interaction of NLRP3 and SLC25A3 is significantly boosted in the mitochondria when the NLRP3 inflammasome is activated. Our detailed investigation demonstrated that the interaction between NLRP3 and SLC25A3 disrupted the interaction of NLRP3-NEK7, promoted ubiquitination of NLRP3, and negatively regulated NLRP3 inflammasome activation. Thus, these findings uncovered a new regulatory mechanism of NLRP3 inflammasome activation, which provides a new perspective for the therapy of NLRP3 inflammasome-associated inflammatory diseases.

The NACHT, leucine-rich repeat, and pyrin domainscontaining protein 3 (collectively known as NLRP3) inflammasome activation plays a critical role in innate immune and pathogenic microorganism infections.However, excessive activation of NLRP3 inflammasome will lead to cellular inflammation and tissue damage, and naturally it must be precisely controlled in the host.Here, we discovered that solute carrier family 25 member 3 (SLC25A3), a mitochondrial phosphate carrier protein, plays an important role in negatively regulating NLRP3 inflammasome activation.We found that SLC25A3 could interact with NLRP3, overexpression of SLC25A3 and knockdown of SLC25A3 could regulate NLRP3 inflammasome activation, and the interaction of NLRP3 and SLC25A3 is significantly boosted in the mitochondria when the NLRP3 inflammasome is activated.Our detailed investigation demonstrated that the interaction between NLRP3 and SLC25A3 disrupted the interaction of NLRP3-NEK7, promoted ubiquitination of NLRP3, and negatively regulated NLRP3 inflammasome activation.Thus, these findings uncovered a new regulatory mechanism of NLRP3 inflammasome activation, which provides a new perspective for the therapy of NLRP3 inflammasome-associated inflammatory diseases.
Solute carrier family 25 member 3 (SLC25A3), the inner mitochondrial membrane phosphate transporter, provides inorganic phosphate to the mitochondrial matrix (30,31).It has been uncovered that decrease of SLC25A3 protein will lead to diminished mitochondrial ATP synthesis rates (30).Therefore, function of SLC25A3 is essential for ATP production in mitochondria.Interestingly, mammalian phosphate carrier protein SLC25A3 can also transport copper both invitro and in vivo (32).Recently, it has been showed that SLC25A3 deletion could induce mitochondrial energy dysfunction drives remodeling of the cardiac mitochondrial protein acylome (33).However, the role of SLC25A3 in regulation of NLRP3 inflammasome activation has not been uncovered.
In this study, we elucidate the mechanism underling suppression of NLRP3 inflammasome activation.The results revealed that SLC25A3 could interact with NLRP3, and overexpression of SLC25A3 negatively regulated NLRP3 inflammasome activation in THP-1-derived macrophages, and knockdown of SLC25A3 could accelerate NLRP3 inflammasome activation in THP-1 derived macrophages and bone marrow-derived macrophages (BMDMs).Moreover, the interaction of NLRP3 and SLC25A3 was significantly enhanced in the mitochondria when NLRP3 inflammasome was activated.The interaction of NLRP3 and SLC25A3 disrupted the interaction of NLRP3-NEK7, promoted ubiquitination of NLRP3, and finally negatively regulated NLRP3 inflammasome activation.Therefore, the work uncovered a distinct mechanism by which SLC25A3 negatively regulates NLRP3 inflammasome activation.

SLC25A3 interacts with NLRP3
In order to reveal the regulatory mechanism of NLRP3 inflammasome activation, we initially screened targeted proteins interacting with NLRP3 by mass spectrometry in HEK293T cells (Fig 1A).Three targeted proteins (SLC25A3,   SLC25A3 negatively regulates NLRP3 inflammasome activation EEF1A1, and EEF1A2) were identified through mass spectrometry (Table 1).The result indicated that SLC25A3 and EEF1A1 could interact with NLRP3, and the interaction between NLRP3 and SLC25A3 is stronger (Fig. 1B).We found that targeted protein SLC25A3 might interact with NLRP3.To reveal if the interaction between NLRP3 and SLC25A3 is reliable, we had conducted different experiments.First, coimmunoprecipitation (Co-IP) confirmed that NLPR3 could interact with SLC25A3 in HEK293T cells (Fig. 1, C-E).Next, glutathione-S-transferase pull-down confirmed that NLRP3 or leucine-rich repeat domain of NLRP3 could interact with SLC25A3 in vitro (Fig. 1, F and G); finally, Co-IP confirmed that nucleotide-binding domain and leucine-rich repeat domains of NLRP3 could interact with SLC25A3 in HEK293T cells (Fig. 1, H and I).The NLRP3 inflammasome comprises NLRP3, ASC, and caspase-1 (15).To reveal the specificity of their interaction between NLRP3 and SLC25A3, we cotransfected SLC25A3 with NLRP3, ASC, or caspase-1 in HEK293T cells, respectively.The result indicated that SLC25A3 could specially interact with NLRP3 (Fig. 1, J and K).Overall, we demonstrated that SLC25A3 could interact with NLRP3.

Overexpression of SLC25A3 could negatively regulate NLRP3 inflammasome activation
To reveal whether SLC25A3 could regulate NLRP3 inflammasome activation, we constructed a reconstructed NLRP3 inflammasome model, in which HEK293T cells were cotransfected with four plasmids encoding NLRP3, ASC, pro-caspase-1, and pro-IL-1β proteins.Reconstructed NLRP3 inflammasome model was constructed successfully by detecting the secretion of mature IL-1β (Fig 2A).IL-1β secretion (Fig. 2B), and mature IL-1β or mature Casp-1 in the supernatant (Fig. 2C) was inhibited by SLC25A3 in reconstructed NLRP3 inflammasome model, and the inhibitory effect is dependent with the plasmid concentration of SLC25A3.To further reveal the role of SLC25A3 in repression of endogenous NLRP3 inflammasome activation, we constructed THP-1 cell lines stably overexpressing SLC25A3.The THP-1 cell lines stably expressing control or SLC25A3 were differentiated into macrophages and stimulated with lipopolysaccharide (LPS) plus nigericin, LPS plus ATP, LPS plus MSU, or LPS plus Alum.IL-1β secretion (Fig. 2D), the release of lactate dehydrogenase (LDH) in the supernatant (Fig. 2E), and mature IL-1β and mature Casp-1 in the supernatant (Fig. 2F) were significantly attenuated by SLC25A3.Thus, we uncovered that SLC25A3 suppressed NLRP3 inflammasome activation.

Knockdown of SLC25A3 could promote NLRP3 inflammasome activation
To further reveal whether SLC25A3 could regulate NLRP3 inflammasome activation, we constructed plasmids stably overexpressing shRNA-control, shRNA-SLC25A3-1, shRNA-SLC25A3-2, or shRNA-SLC25A3-3.HEK293T cells were transfected with these vectors.The result indicated that knockdown effect of shRNA-SLC25A3-2 is the best by RT-PCR (Fig. 3A).THP-1 cell lines stably expressing shRNA-control or shRNA-SLC25A3 were differentiated into macrophages and stimulated with LPS plus nigericin, LPS plus ATP, LPS plus MSU, or LPS plus Alum.IL-1β secretion (Fig. 3B), the release of LDH in the supernatant (Fig. 3C), and mature IL-1β and the cleavage of GSDMD in the supernatant (Fig. 3D) were obviously reduced by SLC25A3.To further uncover the function of SLC25A3 in NLRP3 inflammasome activation in primary cells, we also constructed four vectors (shRNA-control, shRNA-mSLC25A3-1, shRNA-mSLC25A3-2, and mSLC25A3-3).In L929 cells, we validated the effect of these vectors.The result indicated that knockdown effect of shRNA-mSLC25A3-2 is the best (Fig. 3E).BMDMs were infected with lentivirus containing shRNA-control vector or shRNA-mSLC25A3, subsequently stimulated by LPS plus nigericin, LPS plus ATP, LPS plus MSU, and LPS plus Alum.The results indicated that IL-1β secretion (Fig. 3F), and mature IL-1β and mature Casp-1 in the supernatant (Fig. 3G) were also markedly reduced by mSLC25A3.Overall, we found that knockdown of SLC25A3 could promote NLRP3 inflammasome activation.

SLC25A3 specifically suppressed NLRP3 inflammasome activation
To reveal whether SLC25A3 specifically suppressed NLRP3 inflammasome activation, we would explore its function in other inflammasomes (NLRP1, NLRC4, and AIM2 inflammasomes).SLC25A3 could interact with NLRP3, NLRP3, or NLRC4, but not with AIM2 (Fig 4A).We also constructed a reconstructed AIM2/NLRC4/NLRP1 inflammasome model, in which HEK293T cells were cotransfected with four plasmids encoding AIM2/NLRC4/NLRP1, ASC, pro-caspase-1, and pro-IL-1β proteins.The reconstructed AIM2/NLRC4/NLRP1 inflammasome model was successfully constructed by detecting the secretion of mature IL-1β (Fig. S1, A-C).IL-1β secretion was not inhibited by SLC25A3 in reconstructed AIM2/NLRC4/ NLRP1 inflammasome model, and the content of IL-1β secretion was independent with the plasmid concentration of SLC25A3 (Fig. S1, D-F).THP-1 cell lines stably expressing shRNA-control or shRNA-SLC25A3 were differentiated into macrophages and stimulated with LPS plus ATP, LPS plus poly(dA:dT), LPS plus muramyl dipeptide (MDP), or LPS plus Salm.IL-1β secretion (Fig. 4B), and mature IL-1β and mature Casp-1 in the supernatant (Fig. 4C) were obviously reduced by ATP (a stimulator of NLRP3 inflammasome), but not by poly(dA:dT) (a stimulator of AIM2 inflammasome), MDP (a stimulator of NLRP1 inflammasome), or Salm (a stimulator of NLRC4 inflammasome).These results indicated that knockdown of SLC25A3 could specifically promote NLRP3 inflammasome activation.THP-1 cell lines stably expressing plenti-control or plenti-SLC25A3 were differentiated into macrophages and stimulated with LPS plus ATP, LPS plus poly(dA:dT), LPS plus MDP, or LPS plus Salm.IL-1β secretion (Fig. 4D) and mature IL-1β and mature Casp-1 in the supernatant (Fig. 4E) were significantly enhanced by LPS plus ATP, but not by LPS plus poly(dA:dT), LPS plus MDP, or LPS plus Salm.BMDMs were infected with lentivirus containing shRNA-control vector or shRNA-mSLC25A3, subsequently stimulated by LPS plus ATP, LPS plus poly(dA:dT), LPS plus MDP, or LPS plus Salm.The results indicated that IL-1β secretion (Fig. 4F), and mature IL-1β and mature Casp-1 in the supernatant (Fig. 4G) were also markedly enhanced by LPS plus ATP, but not by LPS plus poly(dA:dT), LPS plus MDP, or LPS plus Salm.Knockdown of SLC25A3 could specifically enhance NLRP3 inflammasome activation in BMDMs.However, for the THP-1 cells, poly(dA:dT) and Salm is good to activate AIM2 and NLRC4 inflammasome, MDP was not suitable to activate NLRP1 inflammasome, and it was more widely known as a activator of NOD2.To verify the accuracy of the above results, Vbp (Val-boroPro) was used as a stimulus for NLRP1 inflammasome.Three siRNA-SLC25A3(-1, -2, -3) were identified by RT-PCR in THP-1 cells, and the effect of siRNA-SLC25A3-3 specific to SLC25A3 was the best (Fig. 4H).THP-1 derived macrophages were transfected with siRNA-control or siRNA-SLC25A3-3 and then stimulated with LPS, or LPS plus Val.
The result indicated that knockdown effect of siRNA-SLC25A3-3 in the cells were effective (Fig. 4I).Vbp could indeed activate NLRP1 inflammasome by measuring the content of IL-1β secretion (Fig. 4J).However, knockdown of SLC25A3 could not enhance NLRP1 inflammasome activation stimulated by LPS plus Vbp (Fig. 4J).Overall, we found that SLC25A3 could specifically suppress NLRP3 inflammasome activation.

SLC25A3 negatively regulates NLRP3 inflammasome activation
indicated that knockdown of SLC25A3 promoted ASC oligomerization and the cleavage of GSDMD in the cells (Fig. 5A).Furthermore, the speck formation of endogenous ASC was enhanced by LPS plus ATP in the cells transfected with siRNA-SLC25A3-3 compared to the cells transfected with siRNAcontrol (Fig. 5B).THP-1 cell lines stably expressing plenticontrol or plenti-SLC25A3 were differentiated into macrophages, and subsequently stimulated with LPS plus ATP.The result demonstrated that overexpressing of SLC25A3 promoted ASC oligomerization and the cleavage of GSDMD in the cells (Fig. 5C).Also, the speck formation of endogenous ASC was attenuated by LPS plus ATP in the cells stably expressing plenti-SLC25A3 compared to the cells stably expressing plenti-control (Fig. 5D).Taken together, we found that SLC25A3 suppressed ASC oligomerization mediated by NLRP3 inflammasome.
and inhibit N-terminal GSDMD oligomerization; it could also block pyroptotic cell death and IL-1β release in human monocytes/macrophages (35).We also indicated that NSA could markedly suppress secreted IL-1β induced by LPS plus nigericin, and significantly inhibit the interaction of NLRP3 and SLC25A3 induced by LPS plus nigericin in the THP-1 derived macrophages.Overall, we found that the interaction between SLC25A3 and NLRP3 in the mitochondria was strengthened by NLRP3 inflammasome inducers.
SLC25A3 inhibits the interaction of NLRP3-NEK7 and promotes ubiquitination of NLRP3 How SLC25A3 could negatively regulate NLRP3 inflammasome activation, we explored the deep mechanism by which SLC25A3 regulated the function of NLRP3.Firstly, we identified the interaction between NLRP3 and NEK7.The results indicated that NLRP3 could interact with NEK7 in HEK293T cells and the interaction between NLRP3 and NEK7 could be strengthened by the inducer of NLRP3 inflammasome (ATP) (Fig. 7, A and B).We also found that SLC25A3 could not interact with NEK7 in the HEK293T cells (Fig. 7, C and D).SLC25A3 or NEK7 could competitively bind to NLRP3 and suppress the interaction between NLRP3 and NEK7 or the interaction between NLRP3 and SLC25A3 (Fig. 7, E and F).We also investigated whether the interaction between SLC25A3 and NLRP3 affected the posttranslational modification of NLRP3.In the HEK293T cells, NLRP3 could be ubiquitinated in cells (Fig. 7G).Overexpression of SLC25A3 could promote ubiquitination of NLRP3 in the HEK293T cells (Fig. 7H).To study the stability of NLRP3 protein, we performed a protein decay assay with cycloheximide which blocked cellular protein synthesis.The results showed that overexpression of SLC25A3 had no effect on the stability of NLRP3 protein (Fig. S4).Knockdown of SLC25A3 could suppress ubiquitination of endogenous NLRP3 in THP-1-differentiated macrophages (Fig. 7I).Ubiquitination of NLRP3 always suppressed NLRP3 inflammasome activation (21)(22)(23)(24)(25)(26).That SLC25A3 promoted ubiquitination of NLRP3 and may suppress the activation of NLRP3 inflammasome.These results indicated that SLC25C3 might suppress the activation of NLRP3 inflammasome by disrupting the interaction of NLRP3-NEK7 and promoting ubiquitination of NLRP3.

Discussion
NLRP3 inflammasome is stimulated by pathogen-associated molecular patterns and damage-associated molecular patterns to induce inflammatory responses (36,37).Accurate and tight regulation of NLRP3 inflammasome activation is essential for host cells.It is critical to explore these mechanisms underlying repression of NLRP3 inflammasome activation.We initially showed that SLC25A3 could interact with NLRP3.Subsequently, overexpression of SLC25A3 and knockdown of SLC25A3 could regulate NLRP3 inflammasome activation in macrophages, and the interaction of NLRP3 and SLC25A3 significantly boosted in the mitochondria when NLRP3 inflammasome is activated.Detailed investigation demonstrated that the interaction between NLRP3 and SLC25A3 disrupted the interaction of NLRP3-NEK7, promoted ubiquitination of NLRP3, and finally negatively regulated NLRP3 inflammasome activation.
SLC25A3 is an inner mitochondrial membrane phosphate transporter; our data also indicated that SLC25A3 was located in the mitochondria.SLC25A3 virtually could not interact with NLRP3 without the stimulators of NLRP3 inflammasome in THP-1-derived macrophages.Perhaps, the wide distribution of the endogenous NLRP3 in the cytoplasm could explain this result.Certainly, we found that SLC25A3 could interact with NLRP3 in the HEK293T cells transfected with SLC25A3 and NLRP3 plasmids.This indicated that SLC25A3 could structurally interact with NLRP3.Interestingly, our data indicated that the interaction of NLRP3 and SLC25A3 was significantly enhanced in the mitochondria in the THP-1-derived macrophages stimulated by the stimulator of NLRP3 inflammasome.NLRP3 could be localized to the mitochondria when NLRP3 inflammasome was activated (38,39).Our data also indicated that NLRP3 could be localized to the mitochondria in the THP-1-derived macrophages stimulated by ATP.However, NLRP3 could not interact with SLC25A3 in the space, because SLC25A3 was localized in the mitochondrial inner membrane.It seems unable to explain the interaction of SLC25A3 and NLRP3 in the THP-1-derived macrophages stimulated by ATP.Recently, it has been reported that the mitochondria was damaged early in GSDMD-mediated pyroptosis, and the Nterminal pore-forming GSDMD fragment (GSDMD-NT) caused mitochondrial damage by permeating mitochondrial inner and outer membranes to accelerate and enhance pyroptosis (40,41).The viewpoints seem to explain that the interaction of NLRP3 and SLC25A3 could obviously be enhanced in the THP-1 derived macrophages stimulated by ATP.Damaged mitochondria may provide space for the interaction of NLRP3 and SLC25A3.Exposed SLC25A3 may suppress NLRP3 inflammasome activation by binding unassembled NLRP3.Disulfiram (an inhibitor of GSDMD, inhibits GSDMD pore formation in liposomes) could significantly suppress IL-1β secretion and the interaction of SLC25A3 and NLRP3 in the THP-1 derived macrophages.Acetylcysteine (an inhibitor of reactive oxygen species) treatment had no obvious inhibitory effect on the interaction of SLC25A3-NLRP3 compared to Disulfiram.However, Disulfiram could also block inflammatory TLR4 signaling by targeting MD-2 and its inhibition of TLR4 signaling is independent of GSDMD and caspase-1 (42).To further highlight the role of the inhibitors of GSDMD, we also used NSA rather than Disulfiram as NSA does not inhibit other innate immune pathways such as tolllike receptor signaling and GSDME-mediated cell death (35).The result also indicated NSA could significantly suppress IL-1β secretion and the interaction of SLC25A3 and NLRP3 in the THP-1 derived macrophages.These results support the above hypothesis that NLRP3 could interact with SLC25A3 in the mitochondria by GSDMD.Of course, a large number of experiments will be performed to support our hypothesis.

Cells
L929, HEK293T, and THP-1 cells were purchased from China Center of Type Culture Collection (CCTCC).L929 and HEK293T cells were cultured in Dulbecco's modified Eagle's medium purchased from Gibco supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate.THP-1 cells were cultured in RPMI 1640 purchased from Gibco supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate.THP-1 was induced by phorbol-12-myristate-13-acetate (40 ng/ml) for 12 to 16 h to form THP-1 derived macrophages.BMDMs were isolated from bone marrow of 6 to 8-week-old female mice; these experiments were performed as described previously (24).Briefly, bone marrow was flushed with RPMI 1640 and collected cells were resuspended and passed through a 200-pore sized mesh.The cells were pretreated with Red Blood Cell Lysis Buffer for 5 min, and then the cells were cultured in Dulbecco's modified Eagle's medium and Flag-NEK7.The cell lysates were immunoprecipitated with IgG (Rabbit) or anti-NLRP3 antibodies and then immunoblotted with indicated antibodies.B, THP-1 derived macrophages were primed with LPS (100 ng/ml) for 6 h, then stimulated by ATP (5 mM) at different time points (0 min or 30 min).The cell lysates were immunoprecipitated with IgG or anti-NLRP3 antibody and then immunoblotted with indicated antibodies.C, HEK293T cells were transfected with Vector-Flag and HA-SLC25A3, or Flag-NEK7 and HA-SLC25A3.The cell lysates were immunoprecipitated with anti-Flag antibody and then immunoblotted with indicated antibodies.D, HEK293T cells were transfected with Flag-NEK7 and Vector-HA, or HA-SLC25A3 and Flag-NEK7.The cell lysates were immunoprecipitated with anti-HA antibodies and then immunoblotted with indicated antibodies.E, Flag-NLRP3 and Flag-NEK7 were co-transfected with Vector-HA or HA-SLC25A3 into the HEK293T cells as indicated in the figure.The cell lysates were immunoprecipitated with IgG (Rabbit) or anti-NLRP3 antibodies and then immunoblotted with indicated antibodies.F, Flag-NLRP3 and HA-SLC25A3 were cotransfected with Vector-Flag or Flag-NEK7 into the HEK293T cells as indicated in the figure.The cell lysates were immunoprecipitated with IgG (Rabbit) or anti-NLRP3 antibodies and then immunoblotted with indicated antibodies.G, HEK293T cells were transfected with Flag-NLRP3 and HA-Ub.The cell lysates were immunoprecipitated with anti-Flag antibodies and finally immunoblotted with indicated antibodies.H, HEK293T cells were transfected with Flag-NLRP3, HA-Ub, or Flag-SLC25A3 as indicated in the figure.The cell lysates were immunoprecipitated with anti-NLRP3 antibodies and finally immunoblotted with indicated antibodies.I, THP-1 derived macrophages were transfected with two siRNAs (siRNA-NC and siRNA-SLC25A3-3) and primed with LPS (100 ng/ml) for 6 h.THP-1 derived macrophages then were stimulated with ATP (5 mM, 30 min).The cell lysates were immunoprecipitated with anti-NLRP3 antibodies and finally immunoblotted with indicated antibodies.LPS, lipopolysaccharide; NEK7, NIMA-related kinase-7; NLRP3, NACHT, leucine-rich repeat, and pyrin domain-containing protein 3; SLC25A3, solute carrier family 25 member 3.

Western blot analysis
Firstly, cells were lysed in lyses buffer for Western blot analysis.Cell lysates were separated by 7.5 to 12.5% SDS-PAGE and then transferred onto a nitrocellulose membrane.The membranes were sealed in PBS with 0.1% Tween 20 containing 5% nonfat dried milk for 45 min at room temperature (RT) and then were incubation with first antibodies at 4 C overnight.Next, the membranes were incubated with second antibodies for 45 min at RT. Finally, the membranes were detected with the Western enhanced chemiluminescence substrate (Bio-Rad).

Figure 1 .
Figure1.SLC25A3 interacts with NLRP3.A, the schematic of mass spectrometry.B, HA-NLRP3 was cotransfected with Vector-Flag, Flag-SLC25A3, Flag-EEF1A1, or Flag-EEF1A2 into the HEK293T cells.The cell lysates were immunoprecipitated with anti-Flag antibody, and then immunoblotted with indicated antibodies.C, HEK293T cells were transfected with Flag-SLC25A3 and Vector-HA, Flag-SLC25A3 and HA-NLRP3, or Vector-Flag and HA-NLRP3, respectively.The cell lysates were immunoprecipitated with anti-Flag or anti-HA antibodies and then immunoblotted with indicated antibodies.D, HEK293T cells were transfected with Flag-NLRP3 and Vector-HA, or HA-SLC25A3 and Flag-NLRP3 as indicated in the figure, respectively.The cell lysates were immunoprecipitated with anti-HA antibodies and then immunoblotted with indicated antibodies.E, HEK293T cells were transfected with HA-SLC25A3 and Vector-Flag, or HA-SLC25A3 and Flag-NLRP3 as indicated in the figure, respectively.The cell lysates were immunoprecipitated with anti-Flag antibodies and then immunoblotted with indicated antibodies.F, HEK293T were transfected with HA-SLC25A3.The cell lysates were mixed with GST or GST-LRR, then immunoprecipitated with anti-GST antibodies and finally immunoblotted with indicated antibodies.G, HEK293T cells were transfected with Flag-NLRP3.The cell lysates were mixed with GST or GST-LRR, then immunoprecipitated with anti-GST antibodies and finally immunoblotted with indicated antibodies.H, HEK293T cells were transfected with Flag-SLC25A3 and Vector-HA, HA-NLRP3, HA-Pyrin, HA-NBD, or HA-LRR as indicated in the figure.The cell lysates were immunoprecipitated with anti-HA antibodies and then immunoblotted with indicated antibodies.I, HEK293T cells were transfected with HA-SLC25A3 and Vector-Flag, Flag-NLRP3, Flag-Pyrin, Flag-NBD, or Flag-LRR as indicated in the figure.The cell lysates were immunoprecipitated with anti-Flag antibodies and then immunoblotted with indicated antibodies.J, HEK293T cells were transfected with Flag-SLC25A3 and Vector-HA, HA-NLRP3, HA-ASC, or HA-caspase-1 as indicated in the figure.The cell lysates were immunoprecipitated with anti-HA antibodies, and then immunoblotted with indicated antibodies.K, HEK293T cells were transfected with HA-SLC25A3 and Vector-Flag, Flag-NLRP3, Flag-ASC, or Flag-caspase-1 as indicated in the figure.The cell lysates were immunoprecipitated with anti-Flag antibodies and then immunoblotted with indicated antibodies.GST, glutathione-S-transferase; HA, hemeagglutinin; LRR, leucine-rich repeat; NBD, Nucleotide-binding domain; NLRP3, NACHT, leucine-rich repeat, and pyrin domain-containing protein 3; SLC25A3, solute carrier family 25 member 3.

Figure 7 .
Figure 7. SLC25A3 inhibits the interaction of NLRP3-NEK7 and promotes ubiquitination of NLRP3.A, HEK293T cells were transfected with Flag-NLRP3 and Flag-NEK7.The cell lysates were immunoprecipitated with IgG (Rabbit) or anti-NLRP3 antibodies and then immunoblotted with indicated antibodies.B, THP-1 derived macrophages were primed with LPS (100 ng/ml) for 6 h, then stimulated by ATP (5 mM) at different time points (0 min or 30 min).The cell lysates were immunoprecipitated with IgG or anti-NLRP3 antibody and then immunoblotted with indicated antibodies.C, HEK293T cells were transfected with Vector-Flag and HA-SLC25A3, or Flag-NEK7 and HA-SLC25A3.The cell lysates were immunoprecipitated with anti-Flag antibody and then immunoblotted with indicated antibodies.D, HEK293T cells were transfected with Flag-NEK7 and Vector-HA, or HA-SLC25A3 and Flag-NEK7.The cell lysates were immunoprecipitated with anti-HA antibodies and then immunoblotted with indicated antibodies.E, Flag-NLRP3 and Flag-NEK7 were co-transfected with Vector-HA or HA-SLC25A3 into the HEK293T cells as indicated in the figure.The cell lysates were immunoprecipitated with IgG (Rabbit) or anti-NLRP3 antibodies and then immunoblotted with indicated antibodies.F, Flag-NLRP3 and HA-SLC25A3 were cotransfected with Vector-Flag or Flag-NEK7 into the HEK293T cells as indicated in the figure.The cell lysates were immunoprecipitated with IgG (Rabbit) or anti-NLRP3 antibodies and then immunoblotted with indicated antibodies.G, HEK293T cells were transfected with Flag-NLRP3 and HA-Ub.The cell lysates were immunoprecipitated with anti-Flag antibodies and finally immunoblotted with indicated antibodies.H, HEK293T cells were transfected with Flag-NLRP3, HA-Ub, or Flag-SLC25A3 as indicated in the figure.The cell lysates were immunoprecipitated with anti-NLRP3 antibodies and finally immunoblotted with indicated antibodies.I, THP-1 derived macrophages were transfected with two siRNAs (siRNA-NC and siRNA-SLC25A3-3) and primed with LPS (100 ng/ml) for 6 h.THP-1 derived macrophages then were stimulated with ATP (5 mM, 30 min).The cell lysates were immunoprecipitated with anti-NLRP3 antibodies and finally immunoblotted with indicated antibodies.LPS, lipopolysaccharide; NEK7, NIMA-related kinase-7; NLRP3, NACHT, leucine-rich repeat, and pyrin domain-containing protein 3; SLC25A3, solute carrier family 25 member 3.