apip  (Novus Biologicals)

Journal: Nature Communications

Article Title: APIP regulates the priming of canonical NLRP3 and non-canonical Caspase-11/4 inflammasomes by binding to TRAF6

doi: 10.1038/s41467-025-65893-8

Figure Lengend Snippet: a , b APIP expression in BMDMs from Apip fl/fl (control) and Lyz2-cre; Apip fl/fl ( Apip cKO) mice, untreated (NT) or LPS-stimulated (200 ng/ml, 3 h), analyzed by western blotting ( a ) and qRT-PCR ( b ). c – e IL-1β secretion and caspase-1 activation in control and Apip cKO BMDMs after LPS (200 ng/ml, 3 h) and ATP (3 mM, 30 min) treatment. L + A; LPS + ATP. Supernatants (Sup) and lysates (Cell) were analyzed by western blotting ( c ), and IL-1β ( d ) and caspase-1 ( e ) signals quantified. Data represent mean ± SD ( n = 4 independent cultures; panels b , d , e ). f , g APIP expression in WT and APIP TG/+ BMDMs analyzed by western blotting ( f ) and qRT-PCR ( g ). Data represent mean ± SD ( n = 3 independent cultures). h – j IL-1β secretion and caspase-1 activation in WT and APIP TG/+ BMDMs after LPS (500 ng/ml, 3 h) and ATP (3 mM, 15 min or 30 min) treatment (WT, n = 5; APIP TG/+ , n = 6 independent cultures) ( h , i ). Cells treated with LPS (1 μg/ml, 3 h), and ATP (3 mM, 30 min), and FAM-YVAD-FMK were stained with Hoechst and observed under a fluorescence microscope ( j ). FAM-YVAD-FMK–positive cells were quantified from 4 (NT) or 10 (LPS + ATP) random microscopic fields (technical replicates). Data represent mean ± SD. nd, not detected. k , l Pyroptosis in Apip cKO or APIP TG/+ BMDMs stimulated with LPS and ATP. Control and Apip cKO BMDMs were treated with LPS (200 ng/ml, 3 h) and ATP (3 mM, 1 h) ( n = 4–6 independent cultures; exact n values shown on the graphs/Source Data) ( k ). WT and APIP TG/+ BMDMs were treated with LPS (500 ng/ml, 3 h) and ATP (3 mM, 30 min) ( n = 3 independent cultures) ( l ). Cells were stained with calcein-AM and PI, and PI–positive cells were quantified by fluorescence microscopy. Data represent mean ± SEM. Source data are provided as a Source Data file. Statistical tests: Unpaired two-tailed Student’s t -test ( d , e , g , i ), two-way ANOVA with Holm–Sidak’s ( b ), with Tukey’s ( j ), or with Sidak’s multiple comparison test ( k , l ). ns, non-significant; arb. units, arbitrary units.

Article Snippet: The Apip flox/flox mice were then crossed with Lyz2-Cre mice (stock# 004781, The Jackson Laboratory; generously gifted by Dr. C. Kang, Seoul National University, Korea) to create Lyz2-Cre ; Apip fl/fl ( Apip cKO) mice.

Techniques: Expressing, Control, Western Blot, Quantitative RT-PCR, Activation Assay, Staining, Fluorescence, Microscopy, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: APIP regulates the priming of canonical NLRP3 and non-canonical Caspase-11/4 inflammasomes by binding to TRAF6

doi: 10.1038/s41467-025-65893-8

Figure Lengend Snippet: a – c Stimulatory effect of APIP on ASC oligomerization. Control and Apip cKO BMDMs were untreated (NT) or treated with LPS (200 ng/ml, 3 h) and ATP (3 mM, 30 min) ( a , b ). WT and APIP TG/+ BMDMs were treated with LPS (1 μg/ml, 3 h), Takinib (10 μM, 30 min), and ATP (3 mM, 30 min) ( c ). Cell lysates were incubated with DSS (2 mM, 1 h), and ASC pellets and lysates were analyzed by western blotting ( a , c ). Pellet signals were quantified and normalized to ASC levels in lysates ( b ). Data represent mean ± SD ( n = 3 independent cultures). d Co-IP analysis of the ASC–NLRP3 interaction. WT and APIP TG/+ BMDMs treated as in ( c ) were subjected to Co-IP using an anti-ASC antibody. Input, 5%. e , f Stimulatory effect of APIP on ASC speck formation. Control and Apip cKO BMDMs were treated with LPS (1 μg/ml, 3 h) and ATP (3 mM, 30 min) ( e ). WT and APIP TG/+ BMDMs were treated with LPS (500 ng/ml, 3 h) and ATP (3 mM, 30 min) ( f ). Cells and nuclei were stained with anti-ASC antibody and Hoechst 33342, respectively. ASC speck–positive cells (magenta arrowheads; representative) were quantified from 2 ( f , NT), 4 ( e , NT), or 10 (LPS + ATP) random microscopic fields (technical replicates). Scale bar, 100 μm; nd, not detected. Data represent mean ± SD. Statistical tests: two-way ANOVA with Sidak’s multiple comparison test ( b , e , f ). * p < 0.05, ** p < 0.01, *** p < 0.001. Exact p values are in the Source Data. Source data are provided as a Source Data file. arb. units, arbitrary units.

Article Snippet: The Apip flox/flox mice were then crossed with Lyz2-Cre mice (stock# 004781, The Jackson Laboratory; generously gifted by Dr. C. Kang, Seoul National University, Korea) to create Lyz2-Cre ; Apip fl/fl ( Apip cKO) mice.

Techniques: Control, Incubation, Western Blot, Co-Immunoprecipitation Assay, Staining, Comparison

Journal: Nature Communications

Article Title: APIP regulates the priming of canonical NLRP3 and non-canonical Caspase-11/4 inflammasomes by binding to TRAF6

doi: 10.1038/s41467-025-65893-8

Figure Lengend Snippet: a – f Effect of APIP on non-canonical inflammasome activation. Control and Apip cKO BMDMs ( a – c ), or WT and APIP TG /+ BMDMs ( d – f ) were treated with Pam3CSK4 (1 μg/ml, 4 h) and MCC950 (10 μM, 1 h), and transfected with LPS (2.5 μg/ml, 16 h). Supernatants were analyzed by LDH assay ( a , d ). Cell lysates were analyzed by western blotting ( b , e ), and blot signals quantified by densitometry ( c , f ). LPS (tf), LPS transfection; NT-GSDMD, N-terminal GSDMD. Data represent mean ± SD. ( n = 4 independent cultures; c n = 5). g – l Effect of APIP on caspase-11 expression. Control and Apip cKO BMDMs ( g – i ), or WT and APIP TG /+ BMDMs ( j – l ) were treated with LPS (1 μg/ml, 3 h). Cell lysates were analyzed by western blotting ( g , j ), and signals of pro-CASP11 (p38, arrow) and APIP were quantified by densitometry ( h , i , k , l ). Data represent mean ± SD. ( n = 4 independent cultures; h n = 3). Statistical tests: two-way ANOVA with Tukey’s multiple comparison test ( a , h , k ) or unpaired two-tailed Student’s t -test ( c , d , f , i , l ). ns, non-significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Exact p values are in the Source Data. Source data are provided as a Source Data file. arb. units, arbitrary units.

Article Snippet: The Apip flox/flox mice were then crossed with Lyz2-Cre mice (stock# 004781, The Jackson Laboratory; generously gifted by Dr. C. Kang, Seoul National University, Korea) to create Lyz2-Cre ; Apip fl/fl ( Apip cKO) mice.

Techniques: Activation Assay, Control, Transfection, Lactate Dehydrogenase Assay, Western Blot, Expressing, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: APIP regulates the priming of canonical NLRP3 and non-canonical Caspase-11/4 inflammasomes by binding to TRAF6

doi: 10.1038/s41467-025-65893-8

Figure Lengend Snippet: a – f qRT-PCR analysis of LPS-induced inflammation. J774A.1 cells were transfected with negative control siRNA (NC) or si Apip (100 nM, 48 h) and left untreated (NT) or treated with LPS (500 ng/ml, 3 h) ( a – d ). Control and Apip cKO BMDMs were treated with LPS (200 ng/ml, 3 h) ( e , f ). Total RNAs were analyzed by qRT-PCR. Data represent mean ± SD ( n = 3 independent cultures). g – k Western blot analysis of LPS-induced inflammation. J774A.1 cells were transfected with NC or si Apip (100 nM, 48 h) and treated with LPS (500 ng/ml, 3 h) ( g , h ). RAW264.7 cells were transfected with Mock or APIP for 24 h and treated with LPS (1 μg/ml) alone or with Takinib (10 μM) for 3 h ( i – k ). Cell lysates were analyzed by western blotting ( g , i ), and blot signals quantified by densitometry ( h , j , k ). Data represent mean ± SD ( n = 4 independent cultures; h n = 5 for pro-IL-1β and n = 3 for p-IκBα). Statistical tests: two-way ANOVA with Tukey’s multiple comparison test ( a – d , j ), unpaired two-tailed Student’s t -test ( e , f ), multiple unpaired t -test with Holm–Sidak’s method ( h ), or one-way ANOVA with Tukey’s multiple comparison test ( k ). ns, non-significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Exact p values are in the Source Data. Source data are provided as a Source Data file. arb. units, arbitrary units.

Article Snippet: The Apip flox/flox mice were then crossed with Lyz2-Cre mice (stock# 004781, The Jackson Laboratory; generously gifted by Dr. C. Kang, Seoul National University, Korea) to create Lyz2-Cre ; Apip fl/fl ( Apip cKO) mice.

Techniques: Quantitative RT-PCR, Transfection, Negative Control, Control, Western Blot, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: APIP regulates the priming of canonical NLRP3 and non-canonical Caspase-11/4 inflammasomes by binding to TRAF6

doi: 10.1038/s41467-025-65893-8

Figure Lengend Snippet: a , b Endogenous interaction between APIP and TRAF6 in macrophages. J774A.1 cells left untreated (NT) ( a ) or treated with LPS (1 μg/ml, 10 or 30 min) ( b ), were subjected to Co-IP using an anti-APIP antibody. Input, 5%; arrows, TRAF6; asterisks, non-specific signals. c Requirement of the N-terminal region of APIP for TRAF6 binding. HEK293T cells transfected with APIP WT and deletion (∆) mutants for 24 h (upper) were analyzed by Co-IP using an anti-TRAF6 antibody (lower). Input, 5%; asterisks, light chains. d – f Effect of APIP WT and ΔN60 mutant on pro-IL-1β and p-NF-κB. RAW264.7 cells transfected with Mock, APIP WT, or ΔN60 mutant for 24 h, treated with LPS (500 ng/ml, 3 h), were analyzed by western blotting ( d ). Signals of pro-IL-1β ( e ) and p-NF-κB ( f ) quantified by densitometry. Data represent mean ± SD ( n = 4 independent cultures). g , h Effect of APIP on TRAF6 auto-ubiquitination. RAW264.7 cells transfected with FLAG-TRAF6 alone or with APIP for 24 h ( g ), and J774A.1 cells transfected with NC or si Apip (100 nM, 48 h) ( h ), were treated with MG132 (5 μM, 4 h) and LPS (1 μg/ml, 30 min). Cells were analyzed by Co-IP with anti-FLAG M2 affinity gel ( g ) or anti-TRAF6 antibody ( h ), followed by immunoblotting with anti-ubiquitin (K63, K48, or total) antibodies. Input, 5%. i Inhibition of TRAF6 signaling attenuates APIP-enhanced pyroptosis. WT and APIP TG/+ BMDMs were treated with LPS (500 ng/ml, 3 h), MCC950 (5 μM) or Takinib (20 μM, 30 min), and ATP (3 mM, 1 h). Cells were stained with calcein-AM and PI, and PI–positive cells were quantified. Data represent mean ± SEM ( n = 3–6 independent cultures; exact n values shown on the graph/Source Data) ( i ). Experiments in a – c , g , h were independently repeated three times with similar results. Statistical tests: one-way ANOVA with Tukey’s ( e , f ) or two-way ANOVA with Sidak’s multiple comparison test ( i ). ns, non-significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Exact p values are in the Source Data. Source data are provided as a Source Data file. arb. units, arbitrary units.

Article Snippet: The Apip flox/flox mice were then crossed with Lyz2-Cre mice (stock# 004781, The Jackson Laboratory; generously gifted by Dr. C. Kang, Seoul National University, Korea) to create Lyz2-Cre ; Apip fl/fl ( Apip cKO) mice.

Techniques: Co-Immunoprecipitation Assay, Binding Assay, Transfection, Mutagenesis, Western Blot, Ubiquitin Proteomics, Inhibition, Staining, Comparison

Journal: Nature Communications

Article Title: APIP regulates the priming of canonical NLRP3 and non-canonical Caspase-11/4 inflammasomes by binding to TRAF6

doi: 10.1038/s41467-025-65893-8

Figure Lengend Snippet: a – d Effects of APIP conditional-KO on systemic inflammation. Control and Apip cKO mice (10–11-weeks-old, n = 13 mice per group) were intraperitoneally injected with LPS ( E . coli O111:B4, 15 mg/kg). Survival ( a ) and body temperature ( b ) were monitored. Control and Apip cKO mice (female, 10–12 weeks) were injected with LPS (15 mg/kg, 8 h), and serum IL-1β and TNF were quantified by ELISA ( c , d ). e – h Effects of APIP overexpression on systemic inflammation. WT and APIP TG/+ mice (male, 9 weeks, n = 6 mice per group) were injected with LPS ( E . coli O55:B5, 20 mg/kg). Survival ( e ) and body temperature ( f ) were monitored. WT and APIP TG /+ mice (10–12 weeks) were injected with LPS (15 mg/kg, 8 h) alone or with C25-140 (14 mg/kg, three doses at 12 h intervals, last dose 4 h before LPS) ( g , h ). MCC950 (10 mg/kg) was administered 1 h before and 3.5 h after LPS, followed by 4.5 h incubation ( g ). Serum IL-1β ( g ) and TNF ( h ) were quantified by ELISA. i Serum IL-1β levels in mice co-injected with LPS and MSU. WT, control, and Apip cKO mice (10–12 weeks) were injected with LPS (1.5 mg/kg), followed 4 h later by MSU crystals (50 mg/kg). After 12 h, serum IL-1β was quantified by ELISA. j – m In vivo effects of APIP conditional-KO on bacterial sepsis. Control and Apip cKO mice (10–12 weeks, n = 8 mice) received fecal suspension injection (FSI, 1000 mg/kg). Survival ( j ) and body temperature ( k ) were monitored. Control and Apip cKO mice (10–12 weeks) received FSI (1000 mg/kg, 8 h), and serum IL-1β and TNF were quantified by ELISA ( l , m ). Data, except for survival graphs, represent mean + or ± SD ( n = 3–13 mice per group; exact n values shown on the graphs/Source Data). Source data are provided as a Source Data file. Statistical tests: Log-rank test ( a , e , j ), two-way ANOVA with Sidak’s ( b , f , k ) or Tukey’s multiple comparison test ( g , i ), or unpaired two-tailed Student’s t -test ( c , d , h , l , m ).

Article Snippet: The Apip flox/flox mice were then crossed with Lyz2-Cre mice (stock# 004781, The Jackson Laboratory; generously gifted by Dr. C. Kang, Seoul National University, Korea) to create Lyz2-Cre ; Apip fl/fl ( Apip cKO) mice.

Techniques: Control, Injection, Enzyme-linked Immunosorbent Assay, Over Expression, Incubation, In Vivo, Suspension, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: APIP regulates the priming of canonical NLRP3 and non-canonical Caspase-11/4 inflammasomes by binding to TRAF6

doi: 10.1038/s41467-025-65893-8

Figure Lengend Snippet: APIP binds to TRAF6 and enhances its activity upon LPS exposure. This interaction promotes the NF-κB pathway, driving transcriptional priming of the NLRP3 inflammasome and caspase-11/4 non-canonical inflammasomes, and the JNK pathway, contributing to non-transcriptional priming of the NLRP3 inflammasome. Together, these processes amplify pyroptosis in response to danger signals and exacerbate systemic inflammation in mice. This figure was created in BioRender. Kwangmin (2025) https://BioRender.com/i6tv231 .

Article Snippet: The Apip flox/flox mice were then crossed with Lyz2-Cre mice (stock# 004781, The Jackson Laboratory; generously gifted by Dr. C. Kang, Seoul National University, Korea) to create Lyz2-Cre ; Apip fl/fl ( Apip cKO) mice.

Techniques: Activity Assay

Journal: Nature Communications

Article Title: APIP regulates the priming of canonical NLRP3 and non-canonical Caspase-11/4 inflammasomes by binding to TRAF6

doi: 10.1038/s41467-025-65893-8

Figure Lengend Snippet: a , b APIP expression in BMDMs from Apip fl/fl (control) and Lyz2-cre; Apip fl/fl ( Apip cKO) mice, untreated (NT) or LPS-stimulated (200 ng/ml, 3 h), analyzed by western blotting ( a ) and qRT-PCR ( b ). c – e IL-1β secretion and caspase-1 activation in control and Apip cKO BMDMs after LPS (200 ng/ml, 3 h) and ATP (3 mM, 30 min) treatment. L + A; LPS + ATP. Supernatants (Sup) and lysates (Cell) were analyzed by western blotting ( c ), and IL-1β ( d ) and caspase-1 ( e ) signals quantified. Data represent mean ± SD ( n = 4 independent cultures; panels b , d , e ). f , g APIP expression in WT and APIP TG/+ BMDMs analyzed by western blotting ( f ) and qRT-PCR ( g ). Data represent mean ± SD ( n = 3 independent cultures). h – j IL-1β secretion and caspase-1 activation in WT and APIP TG/+ BMDMs after LPS (500 ng/ml, 3 h) and ATP (3 mM, 15 min or 30 min) treatment (WT, n = 5; APIP TG/+ , n = 6 independent cultures) ( h , i ). Cells treated with LPS (1 μg/ml, 3 h), and ATP (3 mM, 30 min), and FAM-YVAD-FMK were stained with Hoechst and observed under a fluorescence microscope ( j ). FAM-YVAD-FMK–positive cells were quantified from 4 (NT) or 10 (LPS + ATP) random microscopic fields (technical replicates). Data represent mean ± SD. nd, not detected. k , l Pyroptosis in Apip cKO or APIP TG/+ BMDMs stimulated with LPS and ATP. Control and Apip cKO BMDMs were treated with LPS (200 ng/ml, 3 h) and ATP (3 mM, 1 h) ( n = 4–6 independent cultures; exact n values shown on the graphs/Source Data) ( k ). WT and APIP TG/+ BMDMs were treated with LPS (500 ng/ml, 3 h) and ATP (3 mM, 30 min) ( n = 3 independent cultures) ( l ). Cells were stained with calcein-AM and PI, and PI–positive cells were quantified by fluorescence microscopy. Data represent mean ± SEM. Source data are provided as a Source Data file. Statistical tests: Unpaired two-tailed Student’s t -test ( d , e , g , i ), two-way ANOVA with Holm–Sidak’s ( b ), with Tukey’s ( j ), or with Sidak’s multiple comparison test ( k , l ). ns, non-significant; arb. units, arbitrary units.

Article Snippet: For siRNA transfection, J774A.1 and differentiated THP-1 cells were incubated with 100 nM of either negative control siRNA (NC, SN-1002, Bioneer, Korea) or siRNA targeting APIP (J774A.1: si Apip , 56369-2; THP-1: si APIP , 51074-1; primary human macrophages: si APIP #1, 51074-1, si APIP #2, 51074-2; Bioneer, Korea) using Lipofectamine RNAiMAX (13778100, Invitrogen) according to the manufacturer’s protocol.

Techniques: Expressing, Control, Western Blot, Quantitative RT-PCR, Activation Assay, Staining, Fluorescence, Microscopy, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: APIP regulates the priming of canonical NLRP3 and non-canonical Caspase-11/4 inflammasomes by binding to TRAF6

doi: 10.1038/s41467-025-65893-8

Figure Lengend Snippet: a – c Stimulatory effect of APIP on ASC oligomerization. Control and Apip cKO BMDMs were untreated (NT) or treated with LPS (200 ng/ml, 3 h) and ATP (3 mM, 30 min) ( a , b ). WT and APIP TG/+ BMDMs were treated with LPS (1 μg/ml, 3 h), Takinib (10 μM, 30 min), and ATP (3 mM, 30 min) ( c ). Cell lysates were incubated with DSS (2 mM, 1 h), and ASC pellets and lysates were analyzed by western blotting ( a , c ). Pellet signals were quantified and normalized to ASC levels in lysates ( b ). Data represent mean ± SD ( n = 3 independent cultures). d Co-IP analysis of the ASC–NLRP3 interaction. WT and APIP TG/+ BMDMs treated as in ( c ) were subjected to Co-IP using an anti-ASC antibody. Input, 5%. e , f Stimulatory effect of APIP on ASC speck formation. Control and Apip cKO BMDMs were treated with LPS (1 μg/ml, 3 h) and ATP (3 mM, 30 min) ( e ). WT and APIP TG/+ BMDMs were treated with LPS (500 ng/ml, 3 h) and ATP (3 mM, 30 min) ( f ). Cells and nuclei were stained with anti-ASC antibody and Hoechst 33342, respectively. ASC speck–positive cells (magenta arrowheads; representative) were quantified from 2 ( f , NT), 4 ( e , NT), or 10 (LPS + ATP) random microscopic fields (technical replicates). Scale bar, 100 μm; nd, not detected. Data represent mean ± SD. Statistical tests: two-way ANOVA with Sidak’s multiple comparison test ( b , e , f ). * p < 0.05, ** p < 0.01, *** p < 0.001. Exact p values are in the Source Data. Source data are provided as a Source Data file. arb. units, arbitrary units.

Article Snippet: For siRNA transfection, J774A.1 and differentiated THP-1 cells were incubated with 100 nM of either negative control siRNA (NC, SN-1002, Bioneer, Korea) or siRNA targeting APIP (J774A.1: si Apip , 56369-2; THP-1: si APIP , 51074-1; primary human macrophages: si APIP #1, 51074-1, si APIP #2, 51074-2; Bioneer, Korea) using Lipofectamine RNAiMAX (13778100, Invitrogen) according to the manufacturer’s protocol.

Techniques: Control, Incubation, Western Blot, Co-Immunoprecipitation Assay, Staining, Comparison

Journal: Nature Communications

Article Title: APIP regulates the priming of canonical NLRP3 and non-canonical Caspase-11/4 inflammasomes by binding to TRAF6

doi: 10.1038/s41467-025-65893-8

Figure Lengend Snippet: a – f Effect of APIP on non-canonical inflammasome activation. Control and Apip cKO BMDMs ( a – c ), or WT and APIP TG /+ BMDMs ( d – f ) were treated with Pam3CSK4 (1 μg/ml, 4 h) and MCC950 (10 μM, 1 h), and transfected with LPS (2.5 μg/ml, 16 h). Supernatants were analyzed by LDH assay ( a , d ). Cell lysates were analyzed by western blotting ( b , e ), and blot signals quantified by densitometry ( c , f ). LPS (tf), LPS transfection; NT-GSDMD, N-terminal GSDMD. Data represent mean ± SD. ( n = 4 independent cultures; c n = 5). g – l Effect of APIP on caspase-11 expression. Control and Apip cKO BMDMs ( g – i ), or WT and APIP TG /+ BMDMs ( j – l ) were treated with LPS (1 μg/ml, 3 h). Cell lysates were analyzed by western blotting ( g , j ), and signals of pro-CASP11 (p38, arrow) and APIP were quantified by densitometry ( h , i , k , l ). Data represent mean ± SD. ( n = 4 independent cultures; h n = 3). Statistical tests: two-way ANOVA with Tukey’s multiple comparison test ( a , h , k ) or unpaired two-tailed Student’s t -test ( c , d , f , i , l ). ns, non-significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Exact p values are in the Source Data. Source data are provided as a Source Data file. arb. units, arbitrary units.

Article Snippet: For siRNA transfection, J774A.1 and differentiated THP-1 cells were incubated with 100 nM of either negative control siRNA (NC, SN-1002, Bioneer, Korea) or siRNA targeting APIP (J774A.1: si Apip , 56369-2; THP-1: si APIP , 51074-1; primary human macrophages: si APIP #1, 51074-1, si APIP #2, 51074-2; Bioneer, Korea) using Lipofectamine RNAiMAX (13778100, Invitrogen) according to the manufacturer’s protocol.

Techniques: Activation Assay, Control, Transfection, Lactate Dehydrogenase Assay, Western Blot, Expressing, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: APIP regulates the priming of canonical NLRP3 and non-canonical Caspase-11/4 inflammasomes by binding to TRAF6

doi: 10.1038/s41467-025-65893-8

Figure Lengend Snippet: a – f qRT-PCR analysis of LPS-induced inflammation. J774A.1 cells were transfected with negative control siRNA (NC) or si Apip (100 nM, 48 h) and left untreated (NT) or treated with LPS (500 ng/ml, 3 h) ( a – d ). Control and Apip cKO BMDMs were treated with LPS (200 ng/ml, 3 h) ( e , f ). Total RNAs were analyzed by qRT-PCR. Data represent mean ± SD ( n = 3 independent cultures). g – k Western blot analysis of LPS-induced inflammation. J774A.1 cells were transfected with NC or si Apip (100 nM, 48 h) and treated with LPS (500 ng/ml, 3 h) ( g , h ). RAW264.7 cells were transfected with Mock or APIP for 24 h and treated with LPS (1 μg/ml) alone or with Takinib (10 μM) for 3 h ( i – k ). Cell lysates were analyzed by western blotting ( g , i ), and blot signals quantified by densitometry ( h , j , k ). Data represent mean ± SD ( n = 4 independent cultures; h n = 5 for pro-IL-1β and n = 3 for p-IκBα). Statistical tests: two-way ANOVA with Tukey’s multiple comparison test ( a – d , j ), unpaired two-tailed Student’s t -test ( e , f ), multiple unpaired t -test with Holm–Sidak’s method ( h ), or one-way ANOVA with Tukey’s multiple comparison test ( k ). ns, non-significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Exact p values are in the Source Data. Source data are provided as a Source Data file. arb. units, arbitrary units.

Article Snippet: For siRNA transfection, J774A.1 and differentiated THP-1 cells were incubated with 100 nM of either negative control siRNA (NC, SN-1002, Bioneer, Korea) or siRNA targeting APIP (J774A.1: si Apip , 56369-2; THP-1: si APIP , 51074-1; primary human macrophages: si APIP #1, 51074-1, si APIP #2, 51074-2; Bioneer, Korea) using Lipofectamine RNAiMAX (13778100, Invitrogen) according to the manufacturer’s protocol.

Techniques: Quantitative RT-PCR, Transfection, Negative Control, Control, Western Blot, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: APIP regulates the priming of canonical NLRP3 and non-canonical Caspase-11/4 inflammasomes by binding to TRAF6

doi: 10.1038/s41467-025-65893-8

Figure Lengend Snippet: a , b Endogenous interaction between APIP and TRAF6 in macrophages. J774A.1 cells left untreated (NT) ( a ) or treated with LPS (1 μg/ml, 10 or 30 min) ( b ), were subjected to Co-IP using an anti-APIP antibody. Input, 5%; arrows, TRAF6; asterisks, non-specific signals. c Requirement of the N-terminal region of APIP for TRAF6 binding. HEK293T cells transfected with APIP WT and deletion (∆) mutants for 24 h (upper) were analyzed by Co-IP using an anti-TRAF6 antibody (lower). Input, 5%; asterisks, light chains. d – f Effect of APIP WT and ΔN60 mutant on pro-IL-1β and p-NF-κB. RAW264.7 cells transfected with Mock, APIP WT, or ΔN60 mutant for 24 h, treated with LPS (500 ng/ml, 3 h), were analyzed by western blotting ( d ). Signals of pro-IL-1β ( e ) and p-NF-κB ( f ) quantified by densitometry. Data represent mean ± SD ( n = 4 independent cultures). g , h Effect of APIP on TRAF6 auto-ubiquitination. RAW264.7 cells transfected with FLAG-TRAF6 alone or with APIP for 24 h ( g ), and J774A.1 cells transfected with NC or si Apip (100 nM, 48 h) ( h ), were treated with MG132 (5 μM, 4 h) and LPS (1 μg/ml, 30 min). Cells were analyzed by Co-IP with anti-FLAG M2 affinity gel ( g ) or anti-TRAF6 antibody ( h ), followed by immunoblotting with anti-ubiquitin (K63, K48, or total) antibodies. Input, 5%. i Inhibition of TRAF6 signaling attenuates APIP-enhanced pyroptosis. WT and APIP TG/+ BMDMs were treated with LPS (500 ng/ml, 3 h), MCC950 (5 μM) or Takinib (20 μM, 30 min), and ATP (3 mM, 1 h). Cells were stained with calcein-AM and PI, and PI–positive cells were quantified. Data represent mean ± SEM ( n = 3–6 independent cultures; exact n values shown on the graph/Source Data) ( i ). Experiments in a – c , g , h were independently repeated three times with similar results. Statistical tests: one-way ANOVA with Tukey’s ( e , f ) or two-way ANOVA with Sidak’s multiple comparison test ( i ). ns, non-significant. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Exact p values are in the Source Data. Source data are provided as a Source Data file. arb. units, arbitrary units.

Article Snippet: For siRNA transfection, J774A.1 and differentiated THP-1 cells were incubated with 100 nM of either negative control siRNA (NC, SN-1002, Bioneer, Korea) or siRNA targeting APIP (J774A.1: si Apip , 56369-2; THP-1: si APIP , 51074-1; primary human macrophages: si APIP #1, 51074-1, si APIP #2, 51074-2; Bioneer, Korea) using Lipofectamine RNAiMAX (13778100, Invitrogen) according to the manufacturer’s protocol.

Techniques: Co-Immunoprecipitation Assay, Binding Assay, Transfection, Mutagenesis, Western Blot, Ubiquitin Proteomics, Inhibition, Staining, Comparison

Journal: Nature Communications

Article Title: APIP regulates the priming of canonical NLRP3 and non-canonical Caspase-11/4 inflammasomes by binding to TRAF6

doi: 10.1038/s41467-025-65893-8

Figure Lengend Snippet: a – d Effects of APIP conditional-KO on systemic inflammation. Control and Apip cKO mice (10–11-weeks-old, n = 13 mice per group) were intraperitoneally injected with LPS ( E . coli O111:B4, 15 mg/kg). Survival ( a ) and body temperature ( b ) were monitored. Control and Apip cKO mice (female, 10–12 weeks) were injected with LPS (15 mg/kg, 8 h), and serum IL-1β and TNF were quantified by ELISA ( c , d ). e – h Effects of APIP overexpression on systemic inflammation. WT and APIP TG/+ mice (male, 9 weeks, n = 6 mice per group) were injected with LPS ( E . coli O55:B5, 20 mg/kg). Survival ( e ) and body temperature ( f ) were monitored. WT and APIP TG /+ mice (10–12 weeks) were injected with LPS (15 mg/kg, 8 h) alone or with C25-140 (14 mg/kg, three doses at 12 h intervals, last dose 4 h before LPS) ( g , h ). MCC950 (10 mg/kg) was administered 1 h before and 3.5 h after LPS, followed by 4.5 h incubation ( g ). Serum IL-1β ( g ) and TNF ( h ) were quantified by ELISA. i Serum IL-1β levels in mice co-injected with LPS and MSU. WT, control, and Apip cKO mice (10–12 weeks) were injected with LPS (1.5 mg/kg), followed 4 h later by MSU crystals (50 mg/kg). After 12 h, serum IL-1β was quantified by ELISA. j – m In vivo effects of APIP conditional-KO on bacterial sepsis. Control and Apip cKO mice (10–12 weeks, n = 8 mice) received fecal suspension injection (FSI, 1000 mg/kg). Survival ( j ) and body temperature ( k ) were monitored. Control and Apip cKO mice (10–12 weeks) received FSI (1000 mg/kg, 8 h), and serum IL-1β and TNF were quantified by ELISA ( l , m ). Data, except for survival graphs, represent mean + or ± SD ( n = 3–13 mice per group; exact n values shown on the graphs/Source Data). Source data are provided as a Source Data file. Statistical tests: Log-rank test ( a , e , j ), two-way ANOVA with Sidak’s ( b , f , k ) or Tukey’s multiple comparison test ( g , i ), or unpaired two-tailed Student’s t -test ( c , d , h , l , m ).

Article Snippet: For siRNA transfection, J774A.1 and differentiated THP-1 cells were incubated with 100 nM of either negative control siRNA (NC, SN-1002, Bioneer, Korea) or siRNA targeting APIP (J774A.1: si Apip , 56369-2; THP-1: si APIP , 51074-1; primary human macrophages: si APIP #1, 51074-1, si APIP #2, 51074-2; Bioneer, Korea) using Lipofectamine RNAiMAX (13778100, Invitrogen) according to the manufacturer’s protocol.

Techniques: Control, Injection, Enzyme-linked Immunosorbent Assay, Over Expression, Incubation, In Vivo, Suspension, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: APIP regulates the priming of canonical NLRP3 and non-canonical Caspase-11/4 inflammasomes by binding to TRAF6

doi: 10.1038/s41467-025-65893-8

Figure Lengend Snippet: APIP binds to TRAF6 and enhances its activity upon LPS exposure. This interaction promotes the NF-κB pathway, driving transcriptional priming of the NLRP3 inflammasome and caspase-11/4 non-canonical inflammasomes, and the JNK pathway, contributing to non-transcriptional priming of the NLRP3 inflammasome. Together, these processes amplify pyroptosis in response to danger signals and exacerbate systemic inflammation in mice. This figure was created in BioRender. Kwangmin (2025) https://BioRender.com/i6tv231 .

Article Snippet: For siRNA transfection, J774A.1 and differentiated THP-1 cells were incubated with 100 nM of either negative control siRNA (NC, SN-1002, Bioneer, Korea) or siRNA targeting APIP (J774A.1: si Apip , 56369-2; THP-1: si APIP , 51074-1; primary human macrophages: si APIP #1, 51074-1, si APIP #2, 51074-2; Bioneer, Korea) using Lipofectamine RNAiMAX (13778100, Invitrogen) according to the manufacturer’s protocol.

Techniques: Activity Assay