Journal: Pharmacological research

Article Title: TLR4 and AT1R mediate blood-brain barrier disruption, neuroinflammation, and autonomic dysfunction in spontaneously hypertensive rats.

doi: 10.1016/j.phrs.2021.105877

Figure Lengend Snippet: Fig. 1. Progression of MAP elevation in SHRs is dependent upon AT1R and TLR4. Final indirect tail-cuff mean arterial pressure (MAP; mmHg) measure ments of Losartan cohort (n = 6/group) (A) and TAK-242 cohort (n = 11 WKY, 13 SHR, 17 SHR-TAK) (B) at conclusion of respective treatment periods; final direct MAP (mmHg) of TAK-242 sub-cohort (n = 5 WKY, 6 SHR, 10 SHR-TAK; C). Data evaluated by one-way ANOVA with Tukey post-hoc analysis; data shown as mean±SEM; ****p < 0.0001 vs. WKY; +++p < 0.001 vs. SHR; ++++p < 0.0001 vs. SHR.

Article Snippet: Sections were incubated in PBST (0.01 M PBS, 0.1% Triton, 0.04% NaN3) with 5% normal donkey serum and primary antibodies against TLR4 (mouse monoclonal IgG2bκ, 1:250, Novus Biologicals, USA, NB100–56567, lot CJU03–11 and IMG-5031A), ionized calcium-binding adaptor molecule 1 (IBA1; microglial marker [rabbit polyclonal, 1:1000, Wako Chemicals, USA, 019–19742, lot WDK2121 and WDF6884; goat polyclonal, 1:500, Abcam, UK, ab5076, lot GR3195324–1; or rabbit polyclonal, 1:2000, EnCor Biotechnology Inc., USA, RPCA-IBA1, lot 040119]), TNF-α (mouse monoclonal IgG1, 1:100, Santa Cruz Biotechnology, USA, sc-52746, lot C0119), and/or IL-6 (mouse monoclonal IgG2a, 1:100, Santa Cruz Biotechnology, USA, sc32296, lot I2818).

Techniques:

Journal: Pharmacological research

Article Title: TLR4 and AT1R mediate blood-brain barrier disruption, neuroinflammation, and autonomic dysfunction in spontaneously hypertensive rats.

doi: 10.1016/j.phrs.2021.105877

Figure Lengend Snippet: Fig. 2. TLR4 protein expression within CNS cardioregulatory nuclei. Example confocal maximum projection images of PVN vaso pressin (VP; red) and TLR4 (white) from WKY, SHR, SHR-Los, and SHR-TAK (n = 6/group) (A). Percent change in TLR4 staining (% area) compared to WKY in the PVN (B), RVLM (C), and NTS (D). Data evaluated by one-way ANOVA with Tukey post-hoc analysis; shown as mean±SEM; * ** * p < 0.0001 vs. WKY; + ++ + p < 0.0001 vs. SHR; scale bars: 100 µm; 3 V: third ventricle.

Article Snippet: Sections were incubated in PBST (0.01 M PBS, 0.1% Triton, 0.04% NaN3) with 5% normal donkey serum and primary antibodies against TLR4 (mouse monoclonal IgG2bκ, 1:250, Novus Biologicals, USA, NB100–56567, lot CJU03–11 and IMG-5031A), ionized calcium-binding adaptor molecule 1 (IBA1; microglial marker [rabbit polyclonal, 1:1000, Wako Chemicals, USA, 019–19742, lot WDK2121 and WDF6884; goat polyclonal, 1:500, Abcam, UK, ab5076, lot GR3195324–1; or rabbit polyclonal, 1:2000, EnCor Biotechnology Inc., USA, RPCA-IBA1, lot 040119]), TNF-α (mouse monoclonal IgG1, 1:100, Santa Cruz Biotechnology, USA, sc-52746, lot C0119), and/or IL-6 (mouse monoclonal IgG2a, 1:100, Santa Cruz Biotechnology, USA, sc32296, lot I2818).

Techniques: Expressing, Staining

Journal: Cell Reports

Article Title: ERCC6L2 mitigates replication stress and promotes centromere stability

doi: 10.1016/j.celrep.2023.112329

Figure Lengend Snippet: ERCC6L2 is a centromere-associated protein (A) ERCC6L2 is recruited to centromeres. YFP-ERCC6L2 constructs were expressed in U2OS cells, and then fixed and stained against the centromeric marker CENP-A. Scale bar, 5 μm. (B) Schematic representation of ERCC6L2 protein sequence. Truncated ERCC6L2 proteins used in (A), and alignment of ERCC6L2 sequences corresponding to the conserved VIGS domain, are also shown. (C) ERCC6L2 localizes to centromeres throughout interphase. YFP-ERCC6L2 was expressed in U2OS cells, which were subsequently immunostained with specified antibodies. Cyclin A immunostaining was used to distinguish G1 (Cyclin A-negative) from G2 (Cyclin A-positive) cells, while CENP-B was used as a centromeric marker. To identify S-phase cells, YFP-ERCC6L2 transfected cells were pulse labeled with EdU for 15 min and subjected to staining using Click-iT EdU Alexa Fluor 594 imaging kit. Centromeres were detected by CENP-A antibody. Mitotic cells were identified by DAPI-stained condensed chromosomes. Scale bar, 5 μm. (D) Box and whisker plot measuring the effect of the indicated small interfering RNA (siRNA) on formation of CENP-A foci. Cells were transfected with siRNA on 2 consecutive days, and with YFP-ERCC6L2 construct 10 h after first siRNA transfection. They were fixed and stained 48 h following first siRNA transfection. N(cells) > 2,200. (E) Quantification of YFP-ERCC6L2 colocalization with CENP-B in cells transfected with the indicated siRNAs. Conditions as in (D). Bars represent means with standard deviations. N(cells) > 6,000. (F) Box and whisker plot measuring the effect of the indicated siRNA on formation of CENP-B foci. Conditions as in (E). (G) Quantification of YFP-ERCC6L2 colocalization with CENP-A in cells transfected with the indicated siRNAs. Conditions as in (D). Bars represent means with standard deviations. (H) Electrophoretic mobility shift assay with the purified ERCC6L2 1053−1247 fragment. Increasing concentrations of protein (11, 22, 44, 88, 176 nM) were incubated with radioactively labeled single-stranded (left) or double-stranded DNA (right) and resolved by native polyacrylamide gel electrophoresis. Free DNA substrates are marked by green arrows. Nucleoprotein complexes are marked by red arrows. (I) Representative images of metaphase spreads from control and ERCC6L2 −/− hTERT-RPE1 cells. Magnified views of normal and aberrant chromosomal structures are shown in colored frames. Abnormal CO-FISH patterns are marked ( ∗ ). Scale bar, 5 μm. (J) Frequency of aberrant centromere CO-FISH patterns in indicated cell lines. Bars represent means with standard deviations. N(chromosome) ≥ 3,600. Statistics calculated by one-way ANOVA; ∗ p ≤ 0.05. (K) Representative images of centromere aberrations observed by CO-FISH. Magnified views of differentially labeled chromatids are shown in colored frames. Scale bar, 2 μm. (D–G) Statistics calculated by t test assuming unequal variances; ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001, NS not significant.

Article Snippet: ERCC6L2 795−816 aPIP peptide , Genscript , N/A.

Techniques: Construct, Staining, Marker, Sequencing, Immunostaining, Transfection, Labeling, Imaging, Whisker Assay, Small Interfering RNA, Electrophoretic Mobility Shift Assay, Purification, Incubation, Polyacrylamide Gel Electrophoresis, Control

Journal: Cell Reports

Article Title: ERCC6L2 mitigates replication stress and promotes centromere stability

doi: 10.1016/j.celrep.2023.112329

Figure Lengend Snippet: ERCC6L2 deficiency causes disruption of centromeric chromatin (A) QIBC assays measuring CENP-A, CENP-B, and CENP-C intensities in control and ERCC6L2 −/− U2OS cells. Shown is cell cycle distribution of EdU-labeled cells. Individual cells were colored according to the relative CENP-A, CENP-B, and CENP-C intensities, as indicated. N(total cells) > 30,000. (B) Box and whisker plots measuring average intensities of CENP-A, CENP-B, and CENP-C foci in control and ERCC6L2 −/− U2OS cells. N(images) > 250, N(cells) > 30,000. (C) Representative images used for quantifications in (A). Shown are expressions of centromeric proteins, as indicated, with zoomed images of cells in colored frames displaying different levels of focal intensities. Scale bar, 10 μm. (D) ERCC6L2 −/− cells show a reduced CENP-B occupancy. CENP-B ChIP-seq reads mapped to centromeric regions of the representative chromosomes. The tracks show average number of reads (obtained from triplicates in a sample group) covering a given base on the x axis. (E) ERCC6L2 deficiency does not affect expression of centromeric factors CENP-B and CENP-C. Shown are western blots of whole-cell and chromatin extracts derived from control and ERCC6L2 −/− U2OS cells. Blots against histone H3 is used as a loading control. (F) DNA replication measured as intensity of incorporated EdU during the indicated time frames in control and ERCC6L2 −/− U2OS cells. N(images) > 130, N(cells) > 20,000. (G) Quantification of DNA replication in cells exposed to 10 μM Polα inhibitor CD437 during EdU incorporation. N(images) > 130, N(cells) > 20,000. (B), (F), and (G) Box and whisker plots. Statistics calculated by t test assuming unequal variances; ∗∗∗∗ p ≤ 0.0001, NS, not significant.

Article Snippet: ERCC6L2 795−816 aPIP peptide , Genscript , N/A.

Techniques: Disruption, Control, Labeling, Whisker Assay, ChIP-sequencing, Expressing, Western Blot, Derivative Assay

Journal: Cell Reports

Article Title: ERCC6L2 mitigates replication stress and promotes centromere stability

doi: 10.1016/j.celrep.2023.112329

Figure Lengend Snippet: ERCC6L2 regulates centromeric chromatin and alleviates replication stress at genomic repeats (A) Non-proportional Venn diagram illustrating the overlap between the peaks in control and ERCC6L2 −/− triplicates identified by nascent DNA sequencing in U2OS cells. The experiment provides a snapshot of replicated DNA over the duration of BrdU pulse in an asynchronous cell population, and is expected to include regions replicated at different stages of S-phase. Direct comparison between control and ERCC6L2 −/− peaks identifies regions that are, in relative terms, under- or over-replicated in specific genetic backgrounds. Shown are consensus control and ERCC6L2 −/− peaks (common to each genetic background), and unique control and ERCC6L2 −/− peaks (exclusive to each genetic background). (B) Distribution of unique control and ERCC6L2 −/− peaks on chromosome 5 identified by nascent DNA sequencing. Zoomed view of the framed 10-Mb region is shown below. (C) Top recurring motifs among unique control and ERCC6L2 −/− peaks identified by the MEME suite. Shown are p values and numbers of sites. (D) Conservation of the regions surrounding the top recurring motif identified among unique control peaks ( Figure 3 E). Alignment reveals presence of the C-T rich repetitive element upstream of the conserved motif. Secondary structures of selected sequences, predicted by RNA fold server, are shown below. The conserved motif is predicted to constitute a part of a hairpin structure.

Article Snippet: ERCC6L2 795−816 aPIP peptide , Genscript , N/A.

Techniques: Control, DNA Sequencing, Comparison

Journal: Cell Reports

Article Title: ERCC6L2 mitigates replication stress and promotes centromere stability

doi: 10.1016/j.celrep.2023.112329

Figure Lengend Snippet: ERCC6L2 contains an atypical PCNA-binding motif (A) ERCC6L2 colocalizes with PCNA at CENP-C-negative foci. U2OS cells transfected with YFP-ERCC6L2 were stained for CENP-C and PCNA after pre-extraction of soluble proteins. ERCC6L2-and PCNA-positive, but CENP-C negative foci (red arrows) are marked in zoomed images. (B) ERCC6L2 colocalizes with RPA at CENP-C-negative foci. U2OS cells transfected with YFP-ERCC6L2 were stained for CENP-C and RPA. ERCC6L2- and RPA-positive, but CENP-C-negative foci (red arrows) are marked in zoomed images. (C) Colocalization of the C-terminal ERCC6L2 701−1561 fragment with endogenous PCNA. (D) PCNA pull-down with biotinylated peptides. Biotinylated PIP-box and APIM motif peptides were bound to streptavidin beads and incubated with recombinant PCNA. Interactions were assayed by western blotting with PCNA antibody. Wild-type ZRANB3 PIP-box peptide (ZRANB3 PIP) was used as a positive control. Mutant ZRANB3 PIP-box peptide (Q519A, F525A, and F526A, ZRANB3 PIP ∗ ) served as a negative control. ERCC6L2 aPIP and ERCC6L2 aPIP ∗ denote atypical ERCC6L2 peptides (residues 790–811) containing the WT and the Q798A mutant sequences, respectively. (E) Expression patterns of the indicated C-terminal YFP-ERCC6L2 constructs. ERCC6L2 fragments 701–1,098, 728–1,098, and 797–1,098 show patterns reminiscent of replication foci, whereas fragments 820–1,098 and 880–1,098 do not. (F) PCNA pull-down with GST-tagged peptides. Wild-type and Q798A mutant versions of the GST-tagged ERCC6L2 fragments (residues 753–819) were immobilized on GST beads (shown on the left) and incubated with recombinant PCNA. Interactions were assessed by western blotting against PCNA (shown on the right). (G) Schematic representation of different ERCC6L2 fragments tested for formation of PCNA-like foci. A region at the N terminus of fragment 701–1,098 is zoomed below to show sequence alignment of different ERCC6L2 proteins. The conserved atypical PIP-box is marked in a green frame. Targeted mutation sites are indicated by red arrows. (H) Colocalization of YFP-ERCC6L2 wild type, ATPase dead K165 ERCC6L2, and aPIP ∗ mutant (Q798A, C804A, F806A) with CENP-A. N(cells) > 1,400. (I) Colocalization of YFP-ERCC6L2 wild type, ATPase dead K165 ERCC6L2, and aPIP ∗ mutant (Q798A, C804A, F806A) with CENP-B. N(cells) > 1,000. (J) Front and side views of the PCNA ring (white surface and ribbons) with the ERCC6L2 aPIP-box peptide (green sticks). (K) Overview of the hydrogen-bond interaction network between the ERCC6L2 aPIP-box peptide (green) and PCNA (white). Hydrogen bonds are shown as yellow dotted lines. (L) Hydrophobic pocket on PCNA surface (white) with conserved residues that form “hydrophobic plug” (Leu801, Cys804, and Phe806; shown as green sticks) in the ERCC6L2 aPIP-box peptide. (M) Magnified view of the boxed region in (I). (N) Magnified view of the hydrogen-bond interaction network between the ERCC6L2 aPIP-box peptide (green) and PCNA (white, labels in italics). Hydrogen bonds are shown as yellow dotted lines. Intramolecular hydrogen bonds between ERCC6L2 aPIP-box residues are also shown. (H) and (I) Box and whisker plots. Statistics calculated by t test assuming unequal variances; ∗∗∗∗ p ≤ 0.0001. (A), (B), (C), (E) Scale bar, 5 μm.

Article Snippet: ERCC6L2 795−816 aPIP peptide , Genscript , N/A.

Techniques: Binding Assay, Transfection, Staining, Extraction, Incubation, Recombinant, Western Blot, Positive Control, Mutagenesis, Negative Control, Expressing, Construct, Sequencing, Whisker Assay

Journal: Cell Reports

Article Title: ERCC6L2 mitigates replication stress and promotes centromere stability

doi: 10.1016/j.celrep.2023.112329

Figure Lengend Snippet: ERCC6L2 deficiency causes DSB hyper-resection (A) Quantification of ssDNA accumulation using phosphorylated RPA32 (pRPA) as a marker in control and ERCC6L2 −/− U2OS cells. Cells were treated with 50 μg/mL phleomycin for 1 h, and allowed to recover in normal media for 6 h. N(images) > 250, N(cells) > 30,000. (B) Quantification of pRPA levels by western blot. Cells were treated overnight with the indicated doses of phleomycin. pRPA signal, quantified by ImageJ, is expressed as % of total RPA signal, and calculated as an average of three experiments. (C) Quantification of BrdU-labeled ssDNA. Cells were labeled with BrdU for 24 h, treated with 50 μg/mL phleomycin for 1 h, and allowed to recover for 6 h. BrdU immunostaining was performed under native conditions. (D) Quantification of γH2AX foci in cells treated with 50 μg/mL phleomycin for 1 h and allowed to recover in normal media for 6 h. (E) Downregulation of BRCA2 or RAD51 exacerbates pRPA accumulation in and ERCC6L2 −/− cells. Cells were transfected with the indicated siRNAs, treated with 25 μg/mL phleomycin for 1 h, and allowed to recover in normal media for 6 h. (F) Quantification of pRPA foci in control and ERCC6L2 −/− U2OS cells transfected with the indicated siRNAs. Cell treatments were performed as in (E). (G) Downregulation of 53BP1 effector proteins exacerbates pRPA accumulation in ERCC6L2 −/− cells. Cells were transfected with the indicated siRNAs, and treated as in (E). N(images) > 250, N(cells) > 30,000. (A–G) Box and whisker plots. Statistics calculated by t test assuming unequal variances; ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001, NS, not significant.

Article Snippet: ERCC6L2 795−816 aPIP peptide , Genscript , N/A.

Techniques: Marker, Control, Western Blot, Labeling, Immunostaining, Transfection, Whisker Assay

Journal: Cell Reports

Article Title: ERCC6L2 mitigates replication stress and promotes centromere stability

doi: 10.1016/j.celrep.2023.112329

Figure Lengend Snippet: ERCC6L2 deficiency is associated with nuclear abnormalities (A) Categorization of micronuclei based on the presence of centromeric and telomeric signals. Micronuclei can arise as a consequence of chromosome missegregation (centromere- and telomere-positive micronuclei) or chromosome breaks (centromere-positive telomere-negative, centromere-negative telomere-positive, and centromere- and telomere-negative micronuclei). Interstitial fragments (centric, i.e., centromere-positive, or acentric, i.e., centromere-negative) are generated by two DSBs. Terminal fragments are telomere-positive and can be formed by a single DSB. (B) Box and whisker plots measuring micronuclei subtypes in control and ERCC6L2 −/− U2OS cells, as indicated. N(images) > 400, N(cells) > 30,000. (C) Representative images of UFBs stained with PICH, RPA, CENP-C, and FANCD2 antibodies. Cells were exposed to 25 μg/mL phleomycin for 1 h and recovered in VE-821 for 2 days. Scale bar, 5 μm. (D) Representative images of chromosome segregation defects observed in DAPI-stained ERCC6L2 −/− cells. Cell treatments were as in (C). Scale bar, 5 μm. (E) Quantification of UFB subtypes in control and ERCC6L2 −/− U2OS cells. Cell treatments were as in (C). Bars represent means with standard deviations. N(total anaphase cells) > 200. (F) Quantification of chromosome segregation defects in control and ERCC6L2 −/− U2OS cells. Cell treatments were as in (C). Bars represent means with standard deviations. N(total anaphase cells) > 200. (G) CENP-C intensities are not affected by DNA hyper-resection. To minimize the effect of the cell cycle on centromere measurements, cells were arrested in G2 by a 12-h exposure to CDK1 inhibitor RO3306. Shown is the box and whisker plot measuring CENP-C intensities in different subpopulations (in total cells, cells without and with >100 pRPA foci). N(cells) > 50,000. (B), (E–G) Statistics calculated by t test assuming unequal variances; ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001, NS, not significant.

Article Snippet: ERCC6L2 795−816 aPIP peptide , Genscript , N/A.

Techniques: Generated, Whisker Assay, Control, Staining

Journal: Cell Reports

Article Title: ERCC6L2 mitigates replication stress and promotes centromere stability

doi: 10.1016/j.celrep.2023.112329

Figure Lengend Snippet: Roles of ERCC6L2 in DNA replication and at DNA breaks (A) Proposed model of ERCC6L2 role in DNA replication. ERCC6L2 counteracts replication stress at centromeric chromatin (left) and genomic repeats (right). Left: compact chromatin structure at centromeres of ERCC6L2 +/+ cells causes replication slowdown. In contrast, loss of centromeric proteins from α-satellite repeats in ERCC6L2 −/− cells removes the critical epigenetic mark that underlies biological definition of centromeres, ultimately causing “centromere erosion.” Resulting deprotected centromeric DNA is replicated more rapidly, but is not properly assembled into centromeric chromatin. Potentially, deprotected centromeric DNA is also more accessible for operations with non-physiological outcomes, such as centromere fusions. Right: secondary structures that form at genomic repeats impose replication stress, which is alleviated by ERCC6L2 activity in ERCC6L2 +/+ cells. In the absence of ERCC6L2, secondary structures persist and cause replication slowdown. (B) Proposed model of ERCC6L2 role at DNA breaks. In ERCC6L2-proficient cells 53BP1-REV7-Shieldin-CST complex and ERCC6L2 act independently to restrict excessive DNA end resection, and potentiate NHEJ and CSR.

Article Snippet: ERCC6L2 795−816 aPIP peptide , Genscript , N/A.

Techniques: Activity Assay

Journal: Cell Reports

Article Title: ERCC6L2 mitigates replication stress and promotes centromere stability

doi: 10.1016/j.celrep.2023.112329

Figure Lengend Snippet:

Article Snippet: ERCC6L2 795−816 aPIP peptide , Genscript , N/A.

Techniques: Strep-tag, Recombinant, Protease Inhibitor, Transfection, Imaging, Ligation, Mutagenesis, Plasmid Preparation, DNA Sequencing, ChIP-sequencing, Software

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

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