Journal: bioRxiv

Article Title: Structure of zebrafish NLRP3 reveals a novel mode of inflammasome activation

doi: 10.64898/2026.04.17.719140

Figure Lengend Snippet: a , LDH release assay in NLRP3-KO BlaER1 cells reconstituted with the indicated FLAG-tagged NLRP3 constructs or control plasmid. Expression was induced with doxycycline (Dox) for 18 h. Cells were primed with 200 ng/ml LPS for 4h followed by activation with 6.5 µM nigericin, 30 µg/ml imiquimod or 0.025 mg/ml needle toxin for 2 h. Data are represented as mean ± SD, n = 3. b , Western blots of the whole cell lysates from THP-1 NLRP3-KO cells reconstituted with human or zebrafish NLRP3 or zebrafish NLRP3 containing human PYD. Cells were treated with 1 µg/ml LPS for 3 h followed by activation with 20 µM nigericin or 200 µM imiquimod for 1 h. FLAG-tagged NLRP3 constructs and cleaved GSDMD were visualized with corresponding antibodies. c, d, ASC speck formation in HEK293T cells stably expressing YFP-ASC, upon transfection with indicated NLRP3 constructs: human, zebrafish NLRP3 and zebrafish NLRP3 with B30.2-domain deletion (zebrafish NLRP3 ΔB30.2 ) ( c ); wild-type and oligomer-disrupting mutants of zebrafish NLRP3 ( d ). Cells were transfected with the indicated amount of DNA, and the number of ASC specks per image area was calculated 24 h post-transfection. Data are represented as mean ± SD, n = 3.

Article Snippet: Human full-length ASC was cloned into pLenti-EF1a-C-tGFP (Origene, PS100072) and tGFP was exchanged with YFP.

Techniques: Lactate Dehydrogenase Assay, Construct, Control, Plasmid Preparation, Expressing, Activation Assay, Western Blot, Stable Transfection, Transfection

Journal: bioRxiv

Article Title: Structure of zebrafish NLRP3 reveals a novel mode of inflammasome activation

doi: 10.64898/2026.04.17.719140

Figure Lengend Snippet: a , Western blot analysis of NLRP3 expression in reconstituted cell lines in following doxycycline (Dox) induction. Bands corresponding to FLAG-tagged NLRP3 constructs are indicated with arrows. b, c , Western blot analysis of the whole cell lysates from HEK293T cells reconstituted with YFP-ASC and transfected with the indicated NLRP3 constructs, demonstrating relative expression levels of NLRP3 constructs used for ( b ) and ( c ). FLAG-tagged NLRP3 constructs and β-actin were visualized with corresponding antibodies. Cells were transfected with 100 ng DNA per well in a 96-well plate, and the lysates were collected 24 h post-transfection.

Article Snippet: Human full-length ASC was cloned into pLenti-EF1a-C-tGFP (Origene, PS100072) and tGFP was exchanged with YFP.

Techniques: Western Blot, Expressing, Construct, Transfection

Journal: bioRxiv

Article Title: Structure of zebrafish NLRP3 reveals a novel mode of inflammasome activation

doi: 10.64898/2026.04.17.719140

Figure Lengend Snippet: a , Position and sequence of the PYD-FISNA linker in human and zebrafish NLRP3, and zebrafish NLRP3 containing human PYD-FISNA linker sequence encoded by the exon 3 of human NLRP3 gene (red dashed box). Sequences corresponding to FISNA domain and polybasic region are indicated with black lines. Cys130 palmitoylated in human NLRP3 is highlighted in blue. b , SDS-PAGE gels of sucrose gradient fractions of zebrafish NLRP3 WT and zebrafish NLRP3 containing human linker sequence. c , Sequence alignment of zebrafish and human NLRP3. Domains are indicated with lines and color-coded. Residues forming a “face-to-face” interface according to the human (PDB: 7PZC) and mouse (PDB: 7LFH) NLRP3 “cage” structures are indicated with red and orange stars, respectively. Residues forming a “back-to-back” interface according to the human (PDB: 7PZC) and mouse (PDB: 7LFH) NLRP3 “cage” structures are indicated with blue and violet stars, respectively. Numbers correspond to alignment positions within the full-length sequence alignment. d , Position and sequence of known palmitoylation sites in human and zebrafish NLRP3. Palmitoylated residues are highlighted in blue.

Article Snippet: Human full-length ASC was cloned into pLenti-EF1a-C-tGFP (Origene, PS100072) and tGFP was exchanged with YFP.

Techniques: Sequencing, SDS Page

Journal: ACS Omega

Article Title: Peptide Design for Targeting BTB Domain Homodimerization of BACH2: Complementary In Silico and In Vitro Approaches

doi: 10.1021/acsomega.6c01122

Figure Lengend Snippet: Interference of the selected peptides with the interaction of tGFP tagged BACH2 and myc-DDK tagged BACH2, analyzed through coimmunoprecipitation. (A) Full-length (FL) tGFP (turbo GFP) tagged BACH2 and full-length myc-DDK tagged BACH2 were contransfected and overexpressed in HEK293T cells (lane 3). As controls, backbone plasmids pCMV6-AC-GFP (backbone plasmid for tGFP) and pCMV6-Entry (backbone plasmid for MYC-DDK) were similarly subjected to overexpression, and it was confirmed that no proteins of the same molecular weight were observed (lane 2). Additionally, it was confirmed for control purposes that no proteins corresponding to these tags existed in normal HEK293T cells (lane 1). (B) In protein extracts that simultaneously overexpress FL-tGFP BACH2 and FL-myc-DDK tagged BACH2, a control coimmunoprecipitation experiment was implemented to present homodimerization. Immunoprecipitation band was revealed to be antigen-specific through lack of corresponding bands in Dynabeads only and in Dynabeads only isotype control lanes (Above and below immunoblots). Moreover, 55 kDa band was marked to depict the antibody heavy chain that has a molecular weight around 50 kDa (above immunoblot). Overall, signals obtained between 250 and 130 kDa bands indicated that tGFP-tagged FL BACH2 could coimmunoprecipitate with myc-DDK tagged FL BACH2, thus indicating an expected interaction (below immunoblot). HC: heavy chain (antibody heavy chain). (C) After establishing the coimmunoprecipitation system, the peptide interaction phase was initiated. In the first stage, peptide 13 was incubated with the same amount of total protein extract used in coimmunoprecipitation at 0.5 μM level (corresponding to ∼3 times K d ) (left) and at 2.5 μM level (∼16 times K d ) (middle). As the control group, the same volume of PBS was added instead of peptide (PBS only). After peptide incubation, same coimmunoprecipitation procedure shown was applied. As a result of this analyses, it was observed that 0.5 μM peptide 13 was not very effective in disrupting coimmunoprecipitation (left), but at 2.5 μM level, this interaction was completely eliminated (middle). To understand whether the effect of 2.5 μM peptide 13 was comparable with that of control peptide 1, the same experiment was repeated with 2.5 μM peptide 1 (right), and it was observed that this peptide also completely prevented the interaction at this concentration. Rep1 and Rep2 represented experimental duplicates. (D) Since 2.5 μM peptide 13 and control peptide 1 were completely successful in eliminating the coimmunoprecipitation indicating probable interference with BACH2 dimerization, the coimmunoprecipitation experiment was repeated with lower concentrations for peptide 13 and control peptide 1. Thus, the possible difference between peptide 13 and peptide 1 was attempted to be revealed. At this stage, the effect of 1 and 1.5 μM peptide 13 and peptide 1 on tGFP-FL-BACH2/myc-DDK-FL-BACH2 coimmunoprecipitation was investigated. The utilization of 1 and 1.5 μM peptide 13 showed a more effective interference with immunoprecipitation compared with the control peptide 1 at the corresponding concentrations. This information was evaluated as confirming the higher BACH2 affinity of peptide 13 compared with control peptide 1. Rep1 and Rep2 represented experimental duplicates.

Article Snippet: To generate antibody-bound Dynabeads-protein G conjugate, 2 μg anti-tGFP antibody (Origene, Mouse monoclonal turboGFP antibody clone OTI2H8, TA150041) or 5 μg Mouse (G3A1) IgG1 kappa isotype control antibody (Cell Signaling, Mouse (e7Q5L) mAb IgG2b Isotype control, 53484) was incubated with 150 μg Dynabeads-protein G conjugate in 100 μL PBS for 10 min.

Techniques: Plasmid Preparation, Over Expression, Molecular Weight, Control, Immunoprecipitation, Western Blot, Incubation, Concentration Assay

Journal: Genome Biology

Article Title: Herpes simplex virus type 1 R-loops are targets for APOBEC-mediated mutagenesis

doi: 10.1186/s13059-026-04078-y

Figure Lengend Snippet: Orthogonal APOBEC3 overexpression systems link HSV-1 mutagenesis to R-loops across multiple cell types. Schematic of the two complementary gain-of-function approaches used to validate the APOBEC–R-loop connection: a doxycycline-inducible A3A-HA construct in RPE-1 cells and CMV-driven A3A-tGFP or A3G-tGFP in HEK293-AD cells. A Experimental design: following doxycycline (dox) induction, RPE-1 A3A-HA cells were infected with HSV-1 and subjected to DRIP-seq (R-loops), anti-HA ChIP-seq (A3A binding), and viral mutation analysis. B Western blot with anti-HA antibody showing time-dependent accumulation of A3A-HA after doxycycline addition; the 4-h induction point was used for all NGS-based assays. Total protein staining is shown as a loading control. C Representative immunofluorescence image of doxycycline-treated, HSV-1–infected RPE-1 cells, illustrating robust A3A-HA expression (green), viral antigen staining (magenta), and their overlap in merged channel. D Constitutive A3A-tGFP and A3G-tGFP expression in HSV-1–infected HEK293-AD cells. Schematic of CMV-driven A3A-tGFP and A3G-tGFP constructs used to generate stable HEK293-AD lines; these cells were infected with HSV-1 and processed for DRIP-seq, ChIP-seq with anti-tGFP, and mutation profiling. E Live-cell confocal images showing A3A-tGFP and A3G-tGFP distributions in HEK293-AD cells. F Representative genome-browser view of a 300-bp HSV-1 region illustrating APOBEC-type mutations identified in RPE-1 (blue), HEK293-AD (green), and Jurkat (red) cells. Mutations cluster in discrete hotspots that recur across independent experimental platforms. G APOBEC mutation overlap across cell types. Upper panel: Venn diagram of C → T changes in Jurkat, RPE-1 A3A-HA, and HEK293-AD A3A/A3G-tGFP cells, showing a statistically significant shared subset of hotspots, with the strongest overlap between the two epithelial cell systems. Lower panel: Venn diagram comparing C → T mutations in HEK293-AD A3A-tGFP and A3G-tGFP cells. Nearly all C → T mutations in A3G-tGFP cells coincide with those in A3A-tGFP cells, whereas A3A-tGFP generates additional sites not targeted by A3G-tGFP. H Inter-mutation distance analysis. Histograms depict the distribution of distances between adjacent APOBEC-type mutations (blue) compared with an equal number of randomly positioned mutations (yellow) for RPE-1 A3A-HA (top), HEK293-AD A3A-tGFP (middle), and HEK293-AD A3G-tGFP (bottom) cells. In all cases, observed inter-mutation distances are markedly shorter than random (Wilcoxon test p < 2.2 × 10⁻ 1 ⁶), indicating strong clustering of APOBEC edits. I Colocalization of APOBEC binding with R-loops at C → T mutations. Anchor plots show normalized DRIP-seq signal (R-loops, red) and APOBEC3 ChIP-seq signal (A3A-HA and A3A/A3G-tGFP, blue) centered on C → T mutation sites extended by ± 1.5 kb. In RPE-1 cells, the A3A-HA and R-loop peaks coincide, whereas in HEK293-AD cells the A3A/A3G-tGFP signals form twin peaks flanking the central R-loop summits, consistent with APOBEC binding to ssDNA bordering the RNA–DNA hybrid within the broader R-loop domain. J Substitution mutation spectra in APOBEC ChIP and input samples. Table summarizing counts and frequencies of base-substitution types in HSV-1 genomes recovered from A3A/A3G ChIP and matched input DNA. C → T transitions and C → G transversions are enriched compared with other substitution classes (two-proportion z-test p < 0.00001)

Article Snippet: For ChIP, 8 μg of rabbit polyclonal anti-APOBEC3A (D-23) (Sc-130688) and anti-APOBEC3G (H-63) (Sc-48820) rabbit polyclonal antibodies were used (Jurkat cells), while the anti-HA rabbit polyclonal antibody (Abcam ab9110) was used for RPE-1 p dox A3A-HA cells and anti-tGFP mouse monoclonal turboGFP antibody (Origene, clone OTI2H8) for HEK293-AD p CMV A3A-tGFP and p CMV A3G-tGFP cells.

Techniques: Over Expression, Mutagenesis, Construct, Infection, ChIP-sequencing, Binding Assay, Western Blot, Staining, Control, Immunofluorescence, Expressing