Journal: bioRxiv

Article Title: TACE reprograms RANKL-mediated differentiation of macrophages by activating the non-canonical pathway of IRF3

doi: 10.64898/2026.02.11.705073

Figure Lengend Snippet: TACE f/f mice were crossed with myeloid-specific LysM-Cre mice to generate mice lacking TACE in myeloid cells (TACE ΔM ). ( a and b ) BMDMs from TACE con and TACE ΔM mice were stimulated with RANKL (50 ng/ml) for the indicated times. ( a ) Immunoblot analysis using antibodies against phosphorylated ERK (p-ERK), JNK (p-JNK), or p38. ( b ) Nuclear p65 activity was assessed by immunoblot analysis. Lamin B1 was used as a control for nuclear fractions. ( c ) Immunoblot analysis of NFATc1 and α-tubulin during osteoclastogenesis. ( d - i ) BMDMs from TACE and TACE mice were treated with M-CSF and RANKL for two days. Total RNA was analyzed by bulk RNA-seq. ( d ) Schematic diagram of the experimental conditions representing the early phase of RANKL stimulation. ( e ) Volcano plot showing 39 genes upregulated and 118 genes downregulated in TACE cells (|log2 FC| ≥ 2, p < 0.05). ( f ) Gene Ontology (GO) enrichment analysis of the upregulated genes in TACE cells. ( g ) Hallmark pathway analysis of the upregulated genes in TACE cells. ( h ) Expression of IFN-dependent genes CXCL10 and IRF7 was measured by RT-qPCR. ( i ) Upstream regulator analysis was performed using Ingenuity Pathway Analysis (IPA). ( j ) BMDMs from WT mice were stimulated with RANKL (50 ng/ml) for the indicated times. Nuclear and cytosolic proteins were extracted and analyzed by immunoblotting with IRF3, Lamin B1, or α-tubulin antibodies. ( k ) BMDMs from TACE mice were stimulated with RANKL (50 ng/ml) for the indicated times. Nuclear fractions were analyzed by immunoblotting with IRF3 and Lamin B1 antibodies. All data are presented as mean ± SEM. *p < 0.05; **p < 0.01 by one-way ANOVA with post hoc Tukey’s test ( h ). Data represent at least three independent experiments.

Article Snippet: For adenoviral transduction, recombinant adenoviral particles encoding human TACE (ADV-200349) or IRF3 (ADV-212405) and control (1060) adenoviral particles encoding green fluorescent protein (Ad-CMV-GFP) were purchased from Vector Biolabs (Malvern, PA, USA).

Techniques: Western Blot, Activity Assay, Control, RNA Sequencing, Expressing, Quantitative RT-PCR

Journal: bioRxiv

Article Title: TACE reprograms RANKL-mediated differentiation of macrophages by activating the non-canonical pathway of IRF3

doi: 10.64898/2026.02.11.705073

Figure Lengend Snippet: ( a and b ) BMDMs from WT and IRF3 KO mice were cultured with M-CSF and RANKL for 3 days(n=3). ( a ) Efficiency of IRF3 knockout (KO). IRF3 mRNA expression was measured by RT-qPCR and normalized to HPRT. ( b ) Osteoclastogenesis assay. Left: representative images of TRAP-stained cells. Right: quantification of TRAP-positive multinucleated cells as a percentage relative to WT cells. ( c and d ) BMDM from WT mice were transduced with either control-GFP or hIRF3-expressing adenovirus and cultured with M-CSF (20 ng/ml) and RANKL (40 ng/ml) for 3 days(n=3). ( c ) Efficiency of hIRF3. hIRF3 mRNA expression was measured by RT-qPCR and normalized to HPRT ( d ) Osteoclastogenesis assay. Left: TRAP staining of representative images. Scale bar: 100 µm. Right: Quantification of TRAP-positive multinucleated cells. ( e and f ) BMDMs from WT and IRF3 KO mice were nucleofected with either control (CTL) or TACE-targeting siRNAs and cultured with M-CSF. ( e ) Knockdown (KD) efficiency. TACE mRNA levels were measured by qPCR and normalized to HPRT. ( f ) Osteoclastogenesis assay. Left: representative images of TRAP-stained cells. Right: quantification of TRAP-positive multinucleated cells as a percentage relative to WT-NC cells. All data are presented as mean ± SEM. n.s., not significant. Scale bar: 100 µm. *p < 0.05; **p < 0.01 by two-tailed, unpaired t-test ( b , c , d ) or one-way ANOVA with post hoc Tukey’s test ( a , e , f ). Data represent at least three independent experiments.

Article Snippet: For adenoviral transduction, recombinant adenoviral particles encoding human TACE (ADV-200349) or IRF3 (ADV-212405) and control (1060) adenoviral particles encoding green fluorescent protein (Ad-CMV-GFP) were purchased from Vector Biolabs (Malvern, PA, USA).

Techniques: Cell Culture, Knock-Out, Expressing, Quantitative RT-PCR, Staining, Transduction, Control, Knockdown, Two Tailed Test

Journal: bioRxiv

Article Title: TACE reprograms RANKL-mediated differentiation of macrophages by activating the non-canonical pathway of IRF3

doi: 10.64898/2026.02.11.705073

Figure Lengend Snippet: ( a - c ) BMDMs from WT and IRF3 KO mice were treated with M-CSF and RANKL for one day. Total RNA was analyzed by bulk RNA-seq with two biological replicates. ( a ) Volcano plot showing 692 genes upregulated and 262 genes downregulated in IRF3-deficient cells (|log2 FC| ≥ 2, p < 0.05). ( b ) Gene Ontology (GO) enrichment analysis of the upregulated genes in IRF3 KO cells. ( c ) Hallmark pathway analysis of the upregulated genes in IRF3 KO cells. ( d and e ) BMDMs from WT and IRF3 KO mice were stimulated with RANKL (50 ng/ml) for the indicated times(n=3). ( d ) Expression of IFN-dependent genes CXCL10, IRF7, and Mx1 was measured by RT-qPCR. ( e ) Expression of NFATc1 was measured by RT-qPCR as a marker of osteoclastogenesis. All data are presented as mean ± SEM. *p < 0.05; **p < 0.01 by one-way ANOVA with post hoc Tukey’s test ( d , e ).

Article Snippet: For adenoviral transduction, recombinant adenoviral particles encoding human TACE (ADV-200349) or IRF3 (ADV-212405) and control (1060) adenoviral particles encoding green fluorescent protein (Ad-CMV-GFP) were purchased from Vector Biolabs (Malvern, PA, USA).

Techniques: RNA Sequencing, Expressing, Quantitative RT-PCR, Marker

Journal: bioRxiv

Article Title: TACE reprograms RANKL-mediated differentiation of macrophages by activating the non-canonical pathway of IRF3

doi: 10.64898/2026.02.11.705073

Figure Lengend Snippet: ( a – c ) BMDMs from WT and IRF3 KO mice were treated with M-CSF and RANKL for one day, and total RNA was analyzed by RNA sequencing. ( a ) Differential gene expression (DEG) analysis was performed to compare IRF3-dependent genes with those induced by RANKL. ( b ) GSEA analysis of the upregulated genes by IRF3 deficiency in Group C. ( c ) Heatmap of genes in the Hallmark_TNFA signaling pathway. (d) Expression of the Hbegf gene upon RANKL stimulation in WT and IRF3 KO BMDMs was measured by RT-qPCR. ( e ) Expression of the Hbegf gene upon RANKL stimulation in WT and IFNAR KO BMDMs was measured by RT-qPCR. ( f – h ) BMDMs from WT mice were cultured with M-CSF and RANKL in the presence or absence of recombinant HB-EGF (100ng/ml). ( g ) BMDMs from WT mice were stimulated with RANKL (50 ng/ml) for 24 hours. Immunoblotting was performed using NFATc1 and β-actin antibodies. ( h ) Nuclear fractions were analyzed by immunoblotting with IRF3 and Lamin B1 antibodies. ( i ) Schematic representation of TACE-dependent cross-regulation between IRF3 and HB-EGF, which fine-tunes osteoclast differentiation and arthritic bone erosion. All data are presented as mean ± SEM. Scale bar: 100 µm. *p < 0.05; ****p < 0.001 by one-way ANOVA with post hoc Tukey’s test ( d , e ) or two-tailed, unpaired t-test ( f ). Data represent at least three independent experiments.

Article Snippet: For adenoviral transduction, recombinant adenoviral particles encoding human TACE (ADV-200349) or IRF3 (ADV-212405) and control (1060) adenoviral particles encoding green fluorescent protein (Ad-CMV-GFP) were purchased from Vector Biolabs (Malvern, PA, USA).

Techniques: RNA Sequencing, Gene Expression, Expressing, Quantitative RT-PCR, Cell Culture, Recombinant, Western Blot, Two Tailed Test

Journal: Cell Insight

Article Title: HTATSF1 regulates innate antiviral immune response by orchestrating TRAF3-IRF3 and TRAF6-NF-κB pathways

doi: 10.1016/j.cellin.2025.100294

Figure Lengend Snippet: HTATSF1 is important for HSV-1- or SeV-triggered phosphorylation of downstream signaling components. (A) Effects of HTATSF1-deficiency on HSV-1- or SeV-induced phosphorylation of TBK1, IRF3, and STAT1 in THP-1 cells. The control (g NC ) and HTATSF1-deficient (g HTATSF1 ) THP-1 cells (1 × 10 6 ) were left uninfected or infected with HSV-1 (MOI = 1) or SeV (MOI = 1) for the indicated times before immunoblotting analysis with the indicated antibodies. (B) Effects of HTATSF1-deficiency on HSV-1- or SeV-induced phosphorylation of TBK1, IRF3, and STAT1 in BMDMs. Lyz2 -Cre; Htatsf1 fl/fl and Htatsf1 fl/fl BMDM cells (1 × 10 6 ) were left uninfected or infected with HSV-1 (MOI = 1) or SeV (MOI = 1) for the indicated times before immunoblotting analysis with the indicated antibodies.

Article Snippet: Antibodies against HA (TA180128) (OriGene); Flag (F3165) and β-actin (A2228) (Sigma); p-TBK1 S172 (ab109272), TBK1 (ab40676), p-IRF3 S386 (ab76493), TAK1 (ab109526), TRAF6 (ab33915), p65 (ab7970), ubiquitin (ab7254), K48-linkage specific polyubiquitin (ab140601) and K63-linkage specific polyubiquitin (ab179434) (Abcam); IRF3 (sc-33641) and STAT1 (SC-417) (Santa Cruz Biotechnology); Myc (5605), p-IRF3 S396 (4947), p-STAT1 Y701 (9167), TRAF3 (4729), p-TAK1 T184/187 (4508), p-IκBα S32/36 (9246), IκBα (9242), p-IKKα/β S176/177 (2078), IKKβ (2370), and p-p65 S536 (3033) (Cell Signaling Technology), HTATSF1 (20805-1-AP) and HECTD3 (11487-1-AP) (Proteintech) were purchased from the indicated companies.

Techniques: Phospho-proteomics, Control, Infection, Western Blot