Journal: NPJ Precision Oncology

Article Title: IL-11/IL-11R signal inhibition by 9MW3811 remodels immune tumor microenvironment and enhances anti-tumor efficacy of PD-1 blockade

doi: 10.1038/s41698-025-00913-w

Figure Lengend Snippet: A IL-11 is more overexpressed in CHOL, ESCA, HNSC, LUSC, LUAD, COAD, CESC, and BRCA (data from The Cancer Genome Atlas (TCGA) than normal tissues (data from the Genotype-Tissue Expression (GTEx). B Kaplan–Meier survival analysis of IL-11 mRNA levels for BRCA, CESC, CHOL, ACC, LUAD, PAAD, UCS, and COAD with datasets from TCGA and GTEx. C Relative contributions of IL-11 in different cancer types. The scores were evaluated by AIBERT®. D The impact of genetic loss of IL-11Rα on tumor growth was evaluated using MC38 cell line-derived tumor models with IL-11Rα knockout (IL-11Rα KO -MC38). MC38 or IL-11Rα KO -MC38 cells were inoculated subcutaneously into separate groups of C57BL/6J mice. Data are presented as mean ± SEM, n = 8. E The effects of genetic loss of IL-11Rα on tumor growth were assessed in Hepa1-6 tumor models using C57BL/6J wild-type mice and IL-11Rα knockout mice ( IL-11Rα KO -mice ). Hepa1-6 cells were inoculated subcutaneously into separate groups of C57BL/6 J wild-type or IL-11Rα KO mice, Data are presented as mean ± SEM, n = 6.

Article Snippet: IL-11Rα/STAT3-luc HEK293 reporter cell line was constructed by transfecting the expression vector containing human IL-11Rα full-length gene (HG10252-UT, Sino Biological) into STAT3-Luc/HEK293 cells which constitutively express human gp130 STAT3-luciferase (CBPB0002, Nanjing Cobioer).

Techniques: Expressing, Derivative Assay, Knock-Out

Journal: NPJ Precision Oncology

Article Title: IL-11/IL-11R signal inhibition by 9MW3811 remodels immune tumor microenvironment and enhances anti-tumor efficacy of PD-1 blockade

doi: 10.1038/s41698-025-00913-w

Figure Lengend Snippet: A Binding activity of 9MW3811 with recombinant IL-11 from different species. B Binding activity of 9MW3811 with human IL-11, compared with X203, a reported anti-IL-11 neutralizing antibody. C Blocking activity of 9MW3811 against the formation of IL-11/IL-11Rα/gp130 protein complex, compared with X203. D Schematic representation of JAK/STAT3 signaling downstream of IL-11. E JAK/STAT3 signaling transduction inhibitions by 9MW3811 and X203 were performed by luciferase reporter gene assay, using IL-11Rα/STAT3-luc HEK293 cell line. F The inhibition of pSTAT3 stimulated by human IL-11 in HGC-27 tumor cells was detected by immunoblotting assay using Simple Western-Jess. Data is shown as mean± SD.

Article Snippet: IL-11Rα/STAT3-luc HEK293 reporter cell line was constructed by transfecting the expression vector containing human IL-11Rα full-length gene (HG10252-UT, Sino Biological) into STAT3-Luc/HEK293 cells which constitutively express human gp130 STAT3-luciferase (CBPB0002, Nanjing Cobioer).

Techniques: Binding Assay, Activity Assay, Recombinant, Blocking Assay, Transduction, Luciferase, Reporter Gene Assay, Inhibition, Western Blot, Simple Western

Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet:

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Recombinant, Sequencing, Software, Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay

Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet: A, B March5 cKO mice are resistant to P. aeruginosa infection (A) survival rates ( n = 8) and (B) changes in the body weight of March5 fl/fl and March5 fl/fl;Lyz‐Cre ( n = 8) mice after intraperitoneal injection with 1 × 10 7 CFU Pseudomonas aeruginosa . C–F TNF‐α (C), IL‐6 (D), IL‐1β (E) and IL‐18 (F) from serum, spleen homogenate and peritoneal fluid collected from mice ( n = 8) that were sacrificed 12 and 24 h following the bacterial infection. Each cytokine was analyzed by ELISA. Data information: Values, * P < 0.05, ** P < 0.01 (two‐tailed Student's t ‐test or Mantel–Cox test). Data were expressed as the mean ± SEM. See also Fig . Source data are available online for this figure.

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Infection, Injection, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet: A, B (A) Survival rates ( n = 12–14) and (B) variation of body weight of March5 fl/fl and March5 fl/fl;Lyz‐Cre ( n = 7–9) mice after intraperitoneal injection with 28 mg/kg body weight of LPS. C–F ELISA of a TNF‐α (C), IL‐6 (D), IL‐1β (E) and IL‐18 (F) from serum, spleen homogenate and peritoneal fluid from mice ( n = 5), sacrificed 12 and 24 h after LPS injection. Values, * P < 0.05 (two‐tailed Student's t ‐test or Mantel–Cox test.). Data were expressed as the mean ± SEM. Source data are available online for this figure.

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet: A–C (A) Caspase‐1, (B) IL‐1β secretion and (C) LDH release were measured in the supernatants of March5 fl/fl and March5 fl/fl;Lyz‐Cre BMDMs subjected to the indicated stimuli followed by material method. Values are the mean ± SD. Experiments (A–C) were performed in triplicate and repeated at least three times. *** P < 0.001 (two‐tailed Student's t ‐test) D–F MARCH5 mediates activation of the NLRP3 pathway in response to bacterial infection. March5 fl/fl and March5 fl/fl;Lyz‐Cre BMDMs were infected with (D) Citrobacter rodentium (20 MOI, 40 MOI and 80 MOI), (E) Salmonella typhimurium (1 MOI, 5 MOI and 10 MOI), and (F) Pseudomonas aeruginosa (1 MOI, 10 MOI and 20 MOI). Secretion of IL‐1β, IL‐18, IL‐6 and TNF‐α in BMDMs infected for 12 h was measured by ELISA. Values, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (two‐tailed Student's t‐ test). Data are expressed as the mean ± SEM. Experiments (D–F) were performed in triplicate and repeated three times. See also Fig . Source data are available online for this figure.

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Two Tailed Test, Activation Assay, Infection, Enzyme-linked Immunosorbent Assay

Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet: A–C (A) Activated caspase‐1, (B) IL‐1β, and (C) LDH release were measured in the supernatants of siControl (siCtrl) or siMARCH5 THP‐1 cells and were subjected to the indicated stimuli. Independent experiments were repeated at least three times. Values are the mean ± SD. ** P < 0.01, *** P < 0.001 (two‐tailed student's t ‐test). D–G March5 fl/fl and March5 fl/fl;Lyz‐Cre BMDMs were infected with (D) Citrobacter rodentium (20 MOI, 40 MOI and 80 MOI), (E) Salmonella typhimurium (1 MOI, 5 MOI and 10 MOI), and (F) Psedumonas aeruginosa (1 MOI, 10 MOI and 20 MOI). Secretions of caspase‐1 in BMDMs infected for 12 h were measured (D–F), and the cell pellet was used for western blotting to detect the activation of NLRP3 inflammasome (G) in response to bacterial infection. Values, *** P < 0.001, **** P < 0.0001 (two‐tailed Student's t ‐test). Data were expressed as the mean ± SEM. Source data are available online for this figure.

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Two Tailed Test, Infection, Western Blot, Activation Assay

Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet: A MARCH5 WT and KO HEK293T cells were transfected with FLAG‐NLRP3 and HA‐ub. After the cells were stimulated with LPS (200 ng/ml for 4 h) and nigericin (15 μM for 60 min), the cell lysates were immunoprecipitated with FLAG‐M2 beads. Ubiquitinated NLRP3 was detected by anti‐HA antibody. B FLAG‐NLRP3 and HA‐ub K48R mutant plasmids were transfected into MARCH5 KO HEK293T cells with or without Myc‐MARCH5 2KR. Then, the cells were stimulated with 200 ng/ml LPS followed by 15 μM nigericin for the indicated durations. The cell lysates were immunoprecipitated with FLAG‐M2 beads and immunoblotted by using indicated antibodies. C MARCH5 KO HEK293T cells were transfected with FLAG‐NLRP3, Myc‐MARCH5 2KR, and each HA ‐ ubiquitin, shown by the number of the remaining single Lys residue with the other Lys changed to Arg. For stimulation, the cells were treated with 200 ng/ml LPS for 4 h, followed by 15 μM nigericin for 30–60 min. Cell lysates were immunoprecipitated with FLAG‐M2 beads and analyzed by immunoblotting with the indicated antibodies. D The NLRP3 inflammasome was reconstituted in HEK293T MARCH5 KO cells expressing ASC, pro‐caspase‐1, and IL‐1β with HA‐ub WT, K27 only (K27O) mutant, or K27R mutant. Additionally, cells were cotransfected with or without Myc‐MARCH5 2KR or the Myc‐MARCH5 H43W mutant. Following stimulation with LPS for 4 h and nigericin for 30 min, IL‐1β secretion was quantitated by ELISA. Values are the mean ± SD. ** P < 0.01, *** P < 0.001 (two‐tailed Student's t ‐test). All the experiments were carried out in triplicate three times. E HEK293T MARCH5 K/O cells were transfected with FLAG‐NLRP3, Myc‐MARCH5 (2KR), HA‐ubiquitin and GFP‐YOD1. Transfected cells were stimulated with 200 ng/ml LPS for 4 h, followed by 15 μM nigericin for 60 min. Cell lysates were immunoprecipitated with FLAG‐M2 beads and analyzed by ubiquitination with HA‐antibody. The other proteins were detected by indicated antibodies. F Schematic representation of NLRP3 Lys mutants (Upper). HEK293T MARCH5 KO cells were cotransfected with FLAG‐NLRP3 WT or indicated Lys point mutants, HA‐ubiquitin, and Myc‐MARCH5 (2KR). Cells were stimulated with 200 ng/ml LPS for 4 h followed by 15 μM nigericin treatment for 30 min. The cell lysates were immunoprecipitated with FLAG‐M2 beads. NLRP3 ubiquitination was assessed via western blotting using anti‐HA. And each protein was detected by indicated antibodies (Lower). G FLAG‐NLRP3 WT or indicated Lys point mutants, HA‐ub K27 only mutant and Myc‐MARCH5 2KR were transfected into MARCH5 KO HEK293T cells. Cell lysates were immunoprecipitated with FLAG‐M2 beads. Ubiquitinated NLRP3 was detected by immunoblotting using an HA antibody. Representative data are shown from independent experiments that were repeated at least three times. See also Fig . Source data are available online for this figure.

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Transfection, Immunoprecipitation, Mutagenesis, Residue, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet: A March5 fl/fl and March5 fl/fl;Lyz‐Cre BMDMs were primed with LPS for 4 h and were stimulated without or with 5 mM ATP at the indicated time points. Whole‐cell lysates were analyzed via western blotting using the indicated antibodies. B MARCH5 KO HEK293T cells were cotransfected with HA‐ub (K48R) mutant and NLRP3 without or with Myc‐MARCH5 2KR or Myc‐MARCH5 H43W. After treatment with nigericin for 30 min of the LPS treatment for 4 h, the cell lysates were subjected to immunoprecipitation with anti‐FLAG M2 beads. An HA antibody was used to assess NLRP3 ubiquitination. C HEK293T MARCH5 KO cells were cotransfected with HA‐ub (K48R) and NLRP3 WT or NLRP3 truncated mutants with or without SFB‐MARCH5 (2KR). After treatment with LPS and nigericin, the cell lysates were subjected to immunoprecipitation with an anti‐Myc antibody overnight, and NLRP3 ubiquitination was assessed via western blotting by using an anti‐HA antibody. D HEK293T MARCH5 KO cells were transfected HA‐ub (K27O) with NLRP3 WT and truncated mutants. After stimulation with LPS and nigericin, cell lysates were immunoprecipitated with anti‐Myc antibody. The HA antibody detected ubiquitination in the subjects. E The NLRP3 inflammasome was reconstituted in HEK293T MARCH5 KO cells expressing ASC, pro‐caspase 1, and IL‐1β with HA‐ub WT, K27O mutant, or K27R mutant NLRP3. Additionally, cells were cotransfected with or without Myc‐MARCH5 2KR or the Myc‐MARCH5 H43W mutant. After stimulation with LPS for 4 h and nigericin for 30 min, IL‐1β secretion was quantitated using ELISA, and the lysates were detected by western blotting with the indicated antibodies. Source data are available online for this figure.

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Western Blot, Mutagenesis, Immunoprecipitation, Transfection, Expressing, Enzyme-linked Immunosorbent Assay

Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet: A HEK293T 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. B HEK293T 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. C NLRP3 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. D Culture supernatants were obtained from (C) and subjected to ELISA to quantify secreted IL‐1β. 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) E Schematic diagram of NLRP3 inflammasome activation by MARCH5. Upon activation by a priming signal, NLRP3 associates with MAVS on mitochondria ①. MARCH5 recognizes and ubiquitinates two lysine sites, K324 and K430, on the NLRP3 NACHT domain via K27‐linked polyubiquitination ②. NLRP3 ubiquitination by MARCH5 promotes the recruitment of NEK7 and enables self‐oligomerization ③. As a result, ASC and pro‐caspase‐1 are recruited to oligomerized NLRP3 ④ for NLRP3 inflammasome assembly ⑤ and trigger the activation of caspase‐1 and pro‐cytokine cleavage ⑥. Source data are available online for this figure.

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Transfection, Confocal Microscopy, Two Tailed Test, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: The EMBO Journal

Article Title: MARCH5 ‐dependent NLRP3 ubiquitination is required for mitochondrial NLRP3‐NEK7 complex formation and NLRP3 inflammasome activation

doi: 10.15252/embj.2023113481

Figure Lengend Snippet:

Article Snippet: pCMV3‐C‐FLAG‐IL‐1β , Sino Biological , Cat# HG10139‐CF.

Techniques: Recombinant, Sequencing, Software, Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay

Journal: Pediatric Rheumatology Online Journal

Article Title: Deregulation of the IL-1β axis in chronic recurrent multifocal osteomyelitis

doi: 10.1186/1546-0096-12-30

Figure Lengend Snippet: Characterization of PBMCs from CRMO patients. (A-E) Real Time-PCR analysis of gene products involved in the regulation of IL-1β, including ASC , NLRP3 , CASP-1 , IL-1β and of the pro-inflammatory cytokine TNF-α in freshly isolated PBMCs obtained from CRMO patients in remission (gray dots) or with active disease (black dots) and healthy controls (white dots). Values are presented as arbitrary unit (AU). (F-G) IL-1β released in supernatants by PBMCs isolated from CRMO patients in remission or in active disease and from healthy controls, after stimulation with 10 ng/ml LPS for 3 hours (F) or 10 ng/ml LPS for 2 hours followed by treatment with 2 mM ATP for 1 hour (G) . IL-1β was measured by ELISA. Black lines represent the mean value. *p < 0.05, **p < 0.01 vs healthy controls.

Article Snippet: Real-time PCR assays were performed using TaqMan Universal PCR Master mix (Applied Biosystems) and the following gene-expression assays: human IL-1β, TNF-α, CASP-1, ASC and NLRP3 (Applied Biosystem).

Techniques: Real-time Polymerase Chain Reaction, Isolation, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: CARD8 inflammasome mediates pyroptosis of HIV-1-infected cells by sensing viral protease activity

doi: 10.1101/2020.09.25.308734

Figure Lengend Snippet: ( A ) Domain architecture of the CARD8 protein. ( B ) HIV-1 protease cleaves the N-terminus of CARD8. HEK293T cells were transfected with plasmids encoding HA-CARD8 (100ng), together with either the pNL4-3 (1μg) or Pro-D25H (1μg). Cells were collected 24 hours after transfection. Anti-HA, anti-CARD8-N and anti-p24 antibodies were used sequentially on the same blot. ( C ) HIV-1 protease is necessary and sufficient to cleave CARD8. HEK293T cells were transfected with constructs encoding CARD8 (100ng) together with indicated viral plasmids (1μg). Cells were collected 24 hours after transfection. ( D ) RPV enhances HIV-1 protease-mediated cleavage of CARD8. HEK293T cells were transiently transfected with HA-CARD8 (100ng) and indicated viral plasmids (1μg). DMSO or RPV was added 24 hours post transfection. Cell lysates were collected 6 hours after RPV treatment. ( E and F ) HIV-1 protease triggers CASP1-dependent pro-IL-1β processing. HEK293T cells were co-transfected with plasmids encoding CASP1 (2ng), pro-IL-1β (200ng) and an HIV-1 plasmid (1μg). After 24 hours, cells were treated with indicated drugs for another 6 hours. ( G ) RPV induces HIV-1 protease-dependent cleavage of CARD8 in infected cells. HEK293T cells were infected with VSV-G-pseudotyped HIV-1 reporter virus. Infected cells were then transfected with HA-CARD8 (100ng). DMSO or RPV was added 24 hours post transfection. Cell lysates were collected 6 hours after RPV treatment. In B - F , cell lysates were evaluated by immunoblotting. CARD8-FL, full-length CARD8; CARD8-N, N terminal CARD8; free Nt, freed N terminus. Data are representative of three or more independent experiments.

Article Snippet: The IL-1β (HG10139-CM) expression plasmid was purchased from Sino Biological.

Techniques: Transfection, Construct, Plasmid Preparation, Infection, Western Blot

Journal: bioRxiv

Article Title: CARD8 inflammasome mediates pyroptosis of HIV-1-infected cells by sensing viral protease activity

doi: 10.1101/2020.09.25.308734

Figure Lengend Snippet: A list of stop codon mutations or truncations. 1 : All viral plasmids were generated using the NL4-3-ΔEnv-EGFP backbone, except NL4-3/BaL. 2 : NL4-3/BaL has the NL4-3 backbone with EGFP coding sequencing in nef , and the BaL envelop coding sequence. These plasmids were used to transfect HEK293T cells, either for immunoblotting analysis of CARD8 and IL-1β cleavage, or for production of HIV-1 reporter viruses when co-transfected with pVSV-G and lentivirus packaging plasmid. HIV-1 reporter viruses generated using NL4-3-ΔEnv-EGFP backbone are replication-defective. The viral vector NL4-3/BaL can produce replication-competent viruses.

Article Snippet: The IL-1β (HG10139-CM) expression plasmid was purchased from Sino Biological.

Techniques: Generated, Sequencing, Western Blot, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: CARD8 inflammasome mediates pyroptosis of HIV-1-infected cells by sensing viral protease activity

doi: 10.1101/2020.09.25.308734

Figure Lengend Snippet: ( A to E ) HIV-1 protease activation by NNRTIs induced rapid pyroptosis of infected monocytes derived macrophages (MDMs). MDMs were infected with HIV NL4-3/BaL . At day 4, Raltegravir (RAL) and T-20 were added to block new infection. Cells were then treated with RPV, EFV, LPV, or combinations for up to 24 hours. In A to C , GFP + cells were detected by flow cytometry. In D , images of infected MDMs were taken using a Cytation 5 Imaging Multi-Mode Reader (Biotek). In E , culture supernatant was collected for IL-1β ELISA. ( F and G ) Pyroptosis of HIV-1-infected MDMs is CASP1-dependent. MDMs were infected and treated as described above. In F , immunoblot analysis of pro-(p45) and cleaved CASP1 (p10 and p20) in infected MDMs after RPV treatment for 1 hour. In G , infected MDMs were pre-treated with VX-765 (100μM) or Z-VAD-FMK (100μM) for 3 hours and then treated with RPV for 4 hours before flow cytometry analysis. ( H and I ) HIV-1 protease mediated inflammasome activation is proteasome-dependent. MDMs were infected and treated as described above. Infected MDMs were pretreated with proteasome inhibitors MG132, Bort, or Me-Bs for 30 minutes and then treated with RPV for 4 hours. In H , GFP expression was analyzed by flow cytometry. In I , culture supernatant was collected for the detection of IL-1β by ELISA. ( J to I ) The CARD8 inflammasome is required for pyroptosis of HIV-1-infected macrophages. ( J ) knockout of CARD8, ASC, CASP1 or NLRP3 in THP-1 cells was confirmed by immunoblotting. Knockout or control THP-1 cells were infected with VSV-G pseudotyped HIV-1 reporter virus NL4-3-Pol. 3 days after infection, cells were pre-treated with LPS (100ng/ml) for 3 hours before RPV treatment. In K , GFP expression was analyzed by flow cytometry 24 hours post RPV treatment; Data were normalized to the control group. In L , culture supernatant was collected 48 hours post RPV treatment for IL-1β detection. In B, G, H and K , P values were calculated using one-way ANOVA and Turkey multiple comparison tests. In C, E, I and L P values were calculated using two-way ANOVA and Turkey multiple comparison tests. *p < 0.05, ****p < 0.0001. In each bar graph, n≥3. Error bars show mean values with SEM. Data are representative of three or more independent experiments.

Article Snippet: The IL-1β (HG10139-CM) expression plasmid was purchased from Sino Biological.

Techniques: Activation Assay, Infection, Derivative Assay, Blocking Assay, Flow Cytometry, Imaging, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, Knock-Out