Journal: The EMBO Journal

Article Title: FADD is recruited to activated STING oligomers to initiate caspase-mediated NF-κB activation in Drosophila melanogaster

doi: 10.1038/s44318-026-00761-9

Figure Lengend Snippet: ( A ) Cleavage of ectopically expressed Relish C-V5 in WT and Dredd KO S2 cells in response to expression of cGLR1 or PGRP-LC. Dredd KO cells were reconstituted with either WT DREDD N-HA or a catalytically inactive mutant (C386A) as indicated. Representative of n = 2 independent experiments. ( B ) Induction of Sting in yw (control) or yw Dredd−/− flies measured by qPCR 24 h after intrathoracic injection of the STING agonist 3’2’-cGAMP or Tris control. Each data point is derived from a pool of six flies (three male, three female). Bars represent mean ± standard deviation. p values were calculated using a pairwise permutation test corrected with the Benjamini–Hochberg method. ** p = 0.002458 and ns: p = 0.3448. ( C ) Survival of yw (control) or yw Dredd−/− flies injected with DCV and co-injected with 3′2′-cGAMP or Tris control. Points and bars represent mean ± standard error. p values were calculated with a Gehan–Breslow–Wilcoxon test: ** p = 0.0026 and ns: p = 0.0647. ( D ) Cleavage of ectopically expressed Relish C-V5 in WT and Fadd KO S2 cells in response to expression of cGLR1 or PGRP-LC. Fadd KO cells were reconstituted with WT dFADD as indicated. Representative of n = 3 independent experiments. ( E ) Induction of Sting in w 1118 (control) or Fadd −/ − flies measured by qPCR 24 h after intrathoracic injection of 3′2′-cGAMP or Tris control. Each data point is derived from a pool of six flies (three male, three female). Bars represent mean ± standard deviation. p values were calculated using a pairwise permutation test corrected with the Benjamini–Hochberg method. * p = 0.02955 and ns: p = 0.6531. ( F ) Survival of w 1118 (control) or Fadd −/− flies injected with DCV and co-injected with 3′2′-cGAMP or Tris control. Points and bars represent mean ± standard error. p values were calculated with a Gehan–Breslow–Wilcoxon test: * p = 0.0401 and ** p = 0.0024. .

Article Snippet: D. melanogaster Fadd KO flies , Exelixis collection, Harvard Medical School , Fadd f02805.

Techniques: Expressing, Mutagenesis, Control, Injection, Derivative Assay, Standard Deviation

Journal: The EMBO Journal

Article Title: FADD is recruited to activated STING oligomers to initiate caspase-mediated NF-κB activation in Drosophila melanogaster

doi: 10.1038/s44318-026-00761-9

Figure Lengend Snippet: ( A ) Cleavage of ectopically expressed Relish C-V5 in WT or Dredd KO S2 cells in response to expression of cGLR1 or PGRP-LC. Dredd KO cells were reconstituted with either WT DREDD or a catalytically inactive mutant (C386A) at either lower (0.5 ng plasmid) or higher (200 ng plasmid) expression levels. Main Fig. shows a sub-portion of data from this figure. Lysates run on separate gels are indicated by dotted lines. ( B – G ), Induction of Srg1, Srg2 , or Srg3 in yw (control) and yw Dredd-/- flies ( B – D ) or w 1118 (control) and Fadd −/− flies ( E – G ) measured by qPCR 24 h after intrathoracic injection of the STING agonist 3′2′-cGAMP. Each data point is derived from a pool of six flies (three male, three female). Bars represent mean ± standard deviation. P values were calculated using a pairwise permutation test corrected with the Benjamini–Hochberg method: *** p = 0.000734, ** p = 0.001001 ( B , yw), 0.00545 ( B , Dredd −/− ), or 0.005422 ( E ), * p = 0.01087 ( D ), 0.04722 ( E ), 0.01735 ( F ), 0.03859 ( G , w 1118 ), or 0.02056 ( G , Fadd −/− ), ns: p = 0.1291 ( C ), 0.5239 ( D ), or 0.3662 ( F ). .

Article Snippet: D. melanogaster Fadd KO flies , Exelixis collection, Harvard Medical School , Fadd f02805.

Techniques: Expressing, Mutagenesis, Plasmid Preparation, Control, Injection, Derivative Assay, Standard Deviation

Journal: The EMBO Journal

Article Title: FADD is recruited to activated STING oligomers to initiate caspase-mediated NF-κB activation in Drosophila melanogaster

doi: 10.1038/s44318-026-00761-9

Figure Lengend Snippet: ( A ) In silico prediction of the dFADD death domain (DD) (green) binding to the interface of oligomerized dSTING dimers (yellow). The insert shows key interacting residues. ( B ) Induction of Sting reporter in Fadd KO S2 cells upon expression of cGLR1 and reconstitution with WT dFADD or mutants disrupting the predicted dSTING:dFADD interface. ( C ) Induction of the Sting reporter in Sting KO S2 cells upon co-expression of cGLR1 and reconstitution with WT dSTING or mutants disrupting the predicted dSTING:dFADD interface. ( D ) LC/MS -based detection of dFADD peptides from co-immunoprecipitation of ectopically expressed EGFP (negative control) or V5-tagged dSTING in WT S2 cells with or without co-expression of cGAS. Data from n = 2 independent experiments, each containing three biological replicates, are shown. Bars indicate the mean. p values were calculated using two-way ANOVA, corrected with a two-tailed Holm–Šídák post hoc test: **** p < 0.0001, ns = 0.1167. ( E ) Co-immunoprecipitation of dSTING N-HA and dFADD C-V5 ectopically expressed in WT S2 cells with or without co-expression of cGLR1. Representative of n = 2 independent experiments. In B , C data from three independent experiments (different geometrical icons), each performed in biological triplicate, are shown with mean ( n = 9). p values were calculated using two-way ANOVA, corrected with Dunnett’s post hoc test: **** p < 0.0001. .

Article Snippet: D. melanogaster Fadd KO flies , Exelixis collection, Harvard Medical School , Fadd f02805.

Techniques: In Silico, Binding Assay, Expressing, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Negative Control, Two Tailed Test

Journal: The EMBO Journal

Article Title: FADD is recruited to activated STING oligomers to initiate caspase-mediated NF-κB activation in Drosophila melanogaster

doi: 10.1038/s44318-026-00761-9

Figure Lengend Snippet: ( A ) Western blot showing expression of dFADD mutants at high expression levels (200 ng plasmid) to enable detection by immunoblot in contrast to expression levels rescuing signaling in Fadd KO cells (0.5 ng expression plasmid). Lysates run on separate gels are indicated by dotted lines. ( B ) Western blot showing expression of dSTING mutants at high expression levels (750 ng plasmid) to enable detection by immunoblot in contrast to expression levels rescuing signaling in Sting KO cells (10 ng plasmid). ( C – E ) Western blot showing expression of dFADD and DREDD mutants. Mutants were additionally expressed with 200 ng plasmid to verify protein expression. Lysates run on separate gels are indicated by dotted lines. ( F , G ) LC/MS -based detection of dSTING ( F ) or DREDD ( G ) peptides from co-immunoprecipitation of ectopically expressed EGFP (negative control) or V5-tagged dSTING in WT S2 cells with or without co-expression of cGAS. In ( F ), data from n = 2 independent experiments each containing three biological replicates are shown. In ( G ) data from n = 1 independent experiment containing three biological replicates are shown, since unique peptides from DREDD were not confidently detected in the other experiment. Bars indicate the mean. p values were calculated using one-way ANOVA corrected with Tukey’s post hoc test: **** p < 0.0001, *** p = 0.0009, ** p = 0.0011 (EGFP vs dSTING+cGAS) or 0.0031 (dSTING vs dSTING+cGAS), ns: p = 0.4537 ( F ) or 0.5118 ( G ). .

Article Snippet: D. melanogaster Fadd KO flies , Exelixis collection, Harvard Medical School , Fadd f02805.

Techniques: Western Blot, Expressing, Plasmid Preparation, Liquid Chromatography with Mass Spectroscopy, Immunoprecipitation, Negative Control

Journal: The EMBO Journal

Article Title: FADD is recruited to activated STING oligomers to initiate caspase-mediated NF-κB activation in Drosophila melanogaster

doi: 10.1038/s44318-026-00761-9

Figure Lengend Snippet: ( A ) In silico prediction of the dFADD (green) and DREDD (pink) death effector domain (DED) complex. The insert shows key interacting residues. ( B , C ) Activation of Sting reporter upon cGLR1 expression ( B ) or AttA reporter upon PGRP-LC expression ( C ) in Fadd KO S2 cells reconstituted with WT dFADD or a mutant disrupting the predicted dFADD:DREDD interface. Data from three independent experiments (different geometrical icons), each performed in biological triplicate, are shown with mean ( n = 9). p values were calculated using two-way ANOVA, corrected with Dunnett’s post hoc test: **** p < 0.0001, ns: p = 0.6517 ( B ) or 0.5948 ( C ), ( D ) Co-Immunoprecipitation of dFADD and DREDD or indicated mutants expressed in WT S2 cells. Representative of n = 3 independent experiments. ( E , F ) Activation of the Sting reporter upon cGLR1 expression ( E ) or AttA reporter upon PGRP-LC expression ( F ) in Dredd KO S2 cells reconstituted with WT DREDD or mutants disrupting the predicted dFADD:DREDD interface. Data from three independent experiments (different geometrical icons), each performed in biological triplicate, are shown with mean ( n = 9). p values were calculated using two-way ANOVA, corrected with Dunnett’s post hoc test: **** p < 0.0001, ns: 0.9995 ( E ) or 0.1986 ( F ). ( G ) Co-immunoprecipitation of dFADD and DREDD or indicated mutants expressed in WT S2 cells. .

Article Snippet: D. melanogaster Fadd KO flies , Exelixis collection, Harvard Medical School , Fadd f02805.

Techniques: In Silico, Activation Assay, Expressing, Mutagenesis, Immunoprecipitation

Journal: The EMBO Journal

Article Title: FADD is recruited to activated STING oligomers to initiate caspase-mediated NF-κB activation in Drosophila melanogaster

doi: 10.1038/s44318-026-00761-9

Figure Lengend Snippet: ( A ) In silico prediction of the IMD (blue) and dFADD (green) death domain (DD) complex. The insert shows key interacting residues. ( B ) Induction of the AttA reporter in imd KO S2 cells upon expression of PGRP-LC and WT IMD or mutants disrupting the predicted IMD:dFADD interface. ( C ) Co-immunoprecipitation of WT IMD and WT dFADD or indicated IMD mutants ectopically expressed in WT S2 cells. Representative of n = 3 independent experiments. ( D ) Induction of the AttA reporter in Fadd KO S2 cells upon PGRP-LC expression and reconstitution with WT dFADD or indicated mutants disrupting the predicted IMD:dFADD interface. ( E ) Co-immunoprecipitation of WT IMD and WT dFADD or indicated dFADD mutants ectopically expressed in WT S2 cells. Representative of n = 3 independent experiments. ( F ) Induction of the Sting reporter in Fadd KO S2 cells upon expression of cGLR1 and WT dFADD or mutants disrupting the predicted IMD:dFADD interface. In ( B , D , and F ), data from three independent experiments (different geometrical icons), each performed in biological triplicate, are shown with mean ( n = 9). p values were calculated using two-way ANOVA, corrected with Dunnett’s post hoc test: **** p < 0.0001, ns: p = 0.9938. Data in ( D , F ) were derived from the same experiments that are shown in Fig. c, , respectively. .

Article Snippet: D. melanogaster Fadd KO flies , Exelixis collection, Harvard Medical School , Fadd f02805.

Techniques: In Silico, Expressing, Immunoprecipitation, Derivative Assay

Journal: The EMBO Journal

Article Title: FADD is recruited to activated STING oligomers to initiate caspase-mediated NF-κB activation in Drosophila melanogaster

doi: 10.1038/s44318-026-00761-9

Figure Lengend Snippet: ( A ) Western blot showing expression of IMD mutants. Mutants were additionally expressed with 200 ng plasmid to verify protein expression. Lysates run on separate gels are indicated by dotted lines ( n = 1). ( B ) Induction of the AttA reporter in Fadd KO S2 cells upon co-expression of PGRP and reconstitution with WT dFADD or mutants disrupting the predicted dSTING:dFADD interface. Data from three independent experiments (different geometrical icons), each performed in biological triplicate ( n = 9), are shown with mean and bars indicating standard deviation. For each experiment, all measurements were normalized to the mean of WT dFADD + PGRP. P values were calculated using two-way ANOVA, corrected with Dunnett’s post hoc test: **** p < 0.0001. ( C ) DREDD N-HA and dFADD C-V5 or mutants disrupting the dFADD:IMD interface were expressed in S2 cells and immunoprecipitated on anti-V5 beads ( n = 2). ( D ) IMD N-HA and dFADD C-V5 or the R39A/R40A mutant were expressed in S2 cells and immunoprecipitated on anti-V5 beads ( n = 2). .

Article Snippet: D. melanogaster Fadd KO flies , Exelixis collection, Harvard Medical School , Fadd f02805.

Techniques: Western Blot, Expressing, Plasmid Preparation, Standard Deviation, Immunoprecipitation, Mutagenesis

Journal: Clinical and Translational Medicine

Article Title: NLK facilitates Caspase‐8 activation to drive macrophage PANoptosis in sepsis

doi: 10.1002/ctm2.70616

Figure Lengend Snippet: NLK deficiency disrupts PANoptosome assembly and augments RIPK1/3‐ dependent necrosome formation in vivo. (A, B) Representative immunoblots of FADD‐ and RIPK1‐associated complexes, together with corresponding input samples, in macrophages isolated from WT and NKO mice at 8 h post‐CLP. Co‐immunoprecipitates were probed for NLK, Caspase‐8, FADD, RIPK1, p‐RIPK1, RIPK3, and p‐RIPK3. (C–I) Quantification analysis of Caspase‐8, FADD, RIPK1, p‐RIPK1, RIPK3, and p‐RIPK3 in FADD‐associated complexes ( n = 3 independent biological replicates). (J–L) Quantification analysis of p‐RIPK1, RIPK3, and p‐RIPK3 in RIPK1‐associated complexes ( n = 3 independent biological replicates). (M) Representative confocal immunofluorescence images of lung macrophages stained for CD68 (green), Caspase‑8 (red), and ASC (cyan). Merged images indicate ASC–Caspase‐8 colocalisation; boxed regions show magnified views. Scale bars: 50 µm. Statistical significance was determined by using one‑way ANOVA with Bonferroni's post hoc test; * p < .05, ** p < .01 and ns indicates p > .05.

Article Snippet: Antibodies against CD68 (28058‐1‐AP), Caspase‐1 (22915‐1‐AP), p‐RIPK1 (66854‐1‐Ig), FADD ( P14906 ‐1‐AP), HA‐Tag (51064‐2‐AP, 66006‐2‐Ig), Flag‐Tag (66008‐4‐Ig, 20543‐1‐AP), and IgG (B900620) were purchased from Proteintech Group, Inc. (Wuhan, China).

Techniques: In Vivo, Western Blot, Isolation, Immunofluorescence, Staining

Journal: Clinical and Translational Medicine

Article Title: NLK facilitates Caspase‐8 activation to drive macrophage PANoptosis in sepsis

doi: 10.1002/ctm2.70616

Figure Lengend Snippet: NLK deficiency impairs PANoptosome assembly and enhances RIPK1/3‐dependent necrosome formation in macrophages. (A, B) Representative immunoblots of FADD‐ and RIPK1‐associated complexes, together with corresponding input samples, in macrophages isolated from WT and NKO mice at 3 h post‐LPS. Co‐IP were probed for NLK, Caspase‐8, FADD, RIPK1, p‐RIPK1, RIPK3, and p‐RIPK3. (C–I) Quantification analysis of Caspase‐8, FADD, RIPK1, p‐RIPK1, RIPK3, and p‐RIPK3 in FADD‐associated complexes ( n = 3 independent biological replicates). (J–L) Quantification analysis of p‐RIPK1, RIPK3, and p‐RIPK3 in RIPK1‐associated complexes ( n = 3 independent biological replicates). (M) Representative confocal immunofluorescence images showing the co‑localisation of RIPK3 (cyan), ASC (green), and Caspase‑8 (red) in PBS‐ or LPS‐treated BMDMs. Merged images indicate RIPK3–ASC–Caspase‐8 colocalisation; boxed regions show magnified views. Scale bars: 25 µm (merged), 10 µm (zoomed). Statistical differences were analysed by one‑way ANOVA with Bonferroni's post hoc test, * p < .05 and ** p < .01.

Article Snippet: Antibodies against CD68 (28058‐1‐AP), Caspase‐1 (22915‐1‐AP), p‐RIPK1 (66854‐1‐Ig), FADD ( P14906 ‐1‐AP), HA‐Tag (51064‐2‐AP, 66006‐2‐Ig), Flag‐Tag (66008‐4‐Ig, 20543‐1‐AP), and IgG (B900620) were purchased from Proteintech Group, Inc. (Wuhan, China).

Techniques: Western Blot, Isolation, Co-Immunoprecipitation Assay, Immunofluorescence