Journal: bioRxiv

Article Title: Proximity proteomics reveals a role for IFI16 during human coronavirus infection

doi: 10.64898/2026.04.22.720112

Figure Lengend Snippet: IFI16 promotes coronavirus infection. ( a) CRISPR-Cas9 technology was used to knock out IFI16 in A549 cells and single cell clones were screened for loss of IFI16 expression using qRT-PCR. Data is representative of two independent experiments. ( b) Immunoblot showing the loss of IFI16 expression in IFI16-KO cell line. ( c) Immunofluorescence assay showing reduced number of HCoV-OC43 N-positive cells upon loss of IFI16. Cells were infected at an MOI of 0.1 and incubated for the indicated length of time before fixation and staining for HCoV-OC43 N protein. Scale bar is 10 um. ( d ) Representative flow cytometry analysis of wildtype (WT) A549 cells and IFI16-KO cells infected (in quadruplicate) with HCoV-OC43 at an MOI of 0.01 and 0.1 for 12, 24 and 48 h. Data is representative of 3 independent experiments. (e) and (f) Quantification of flow cytometry data in . ( g) Representative flow cytometry analysis of CRISPRi cell lines infected (in quadruplicate) with HCoV-OC43 at an MOI of 0.01 for 12, 24 and 48 h. data in figure. (h) Quantification of flow cytometry data in . Data is representative of two independent experiments. Statistical significance was assessed by ANOVA. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001.

Article Snippet: The Taqman assay for IFI16 (Applied Biosystems, ID: Hs00986757_m1) was employed in the detection of IFI16 expression following the manufacturers protocol.

Techniques: Infection, CRISPR, Knock-Out, Single Cell, Clone Assay, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Incubation, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: Proximity proteomics reveals a role for IFI16 during human coronavirus infection

doi: 10.64898/2026.04.22.720112

Figure Lengend Snippet: IFI16 enhances coronavirus replication. (a) WT and IFI16-KO cells were infected with HCoV-OC43 virus (MOI = 0.1) and RNA was harvested at indicated timepoints post infection for qRT-PCR analysis of N gene levels (n = 2 biologically independent samples). Data is representative of two independent experiments. (b) WT and IFI16-KO cells were infected with HCoV-OC43 virus (MOI = 0.1) and cells harvested at indicated timepoints post infection to stain for N protein. Flow cytometry was carried out to quantify the number of virus infected cells at each time point (n = 3 biologically independent samples). Data is representative of two independent experiments. (c) WT and IFI16-KO cells were infected with HCoV-OC43 virus (MOI = 1) and RNA was harvested at different times post infection for qRT-PCR analysis of N gene levels (n = 2 biologically independent samples). Data is representative of two independent experiments. (d) WT and IFI16-KO cells were infected with HCoV-OC43 virus (MOI = 1) and cells harvested at different timepoints post infection to stain for N protein. Flow cytometry was carried out to quantify the number of virus infected cells at each time point. (n = 4 biologically independent samples). Data is representative of two independent experiments. Statistical significance was assessed by ANOVA. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001.

Article Snippet: The Taqman assay for IFI16 (Applied Biosystems, ID: Hs00986757_m1) was employed in the detection of IFI16 expression following the manufacturers protocol.

Techniques: Infection, Virus, Quantitative RT-PCR, Staining, Flow Cytometry

Journal: bioRxiv

Article Title: Proximity proteomics reveals a role for IFI16 during human coronavirus infection

doi: 10.64898/2026.04.22.720112

Figure Lengend Snippet: The role of IFI16 in coronavirus replication is independent of its role as a transcriptional regulator of type I IFN genes. WT and IFI16-KO cells were infected with HCoV-OC43 virus (MOI = 0.1) and RNA was harvested at 12 and 24hr post infection for qRT-PCR analysis of the expression levels of type I IFNs ( a and b ) and RIG-I ( c ). Data is representative of two independent experiments (n = 3 biologically independent samples). (d) WT and IFI16-KO cells were stimulated with poly I:C for 24 h and then infected with HCoV-OC43 virus (MOI = 1) for 12, 24 and 48 h. Cells were harvested and stained for N protein, followed by flow cytometry. Statistical significance was assessed by ANOVA. * P ≤ 0.05, *** P ≤ 0.001, and **** P ≤ 0.0001

Article Snippet: The Taqman assay for IFI16 (Applied Biosystems, ID: Hs00986757_m1) was employed in the detection of IFI16 expression following the manufacturers protocol.

Techniques: Infection, Virus, Quantitative RT-PCR, Expressing, Staining, Flow Cytometry

Journal: Cell Death & Disease

Article Title: IFI16 is essential to linking DNA damage and ferroptosis in acute kidney injury

doi: 10.1038/s41419-026-08604-5

Figure Lengend Snippet: A Western blot analysis showing the expression of GFP-tagged IFI16 and IFI16 bearing PYRIN or HINA&B domain deletions in sgKO-IFI16 HK-2 cells transduced with lentivirus coding wild-type IFI16-GFP, IFI16-ΔPYRIN-GFP mutant, or IFI16-ΔHINA&B-GFP mutant. An anti-GFP antibody was used to detect GFP-tagged proteins. IFI16 antibody (1G7) targeting the N-terminus (1-159aa) was used to detect endogenous IFI16, wild-type IFI16-GFP, and IFI16-ΔHINA&B-GFP mutant. IFI16 antibody targeting the C-terminus (580-729aa) was used to detect endogenous IFI16, wild-type IFI16-GFP, and IFI16-ΔPYRIN-GFP mutant. B Representative photomicrographs and quantification of γ-H2AX immunofluorescence staining in different IFI16-mutant HK-2 cells with H/R 2 h treatment ( n = 8). C Representative Western blot gel documents showing the protein levels of p-ATM, p-KAP1, p-NBS1, and p-p53 in different IFI16-mutant HK-2 cells with H/R 2 h treatment. D Quantitative analysis of MDA levels in different IFI16-mutant HK-2 cells with H/R 24 h treatment ( n = 6). Representative photomicrographs ( E ) and quantification ( F ) of FerroOrange staining in different IFI16-mutant HK-2 cells with H/R 24 h treatment ( n = 8). Representative photomicrographs ( G ) and quantification ( H ) of propidium iodide (PI) staining in different IFI16-mutant HK-2 cells with H/R 24 h treatment ( n = 8). I The extracellular LDH levels in different IFI16-mutant HK-2 cells with H/R 24 h treatment ( n = 6). Data are represented as the mean ± SD. *** p < 0.001, ns not significant.

Article Snippet: IFI16 ( NM_005531 (samesense mut)), IFI16-ΔPYRIN ( NM_005531 (del1-87aa)) and IFI16-ΔHINA&B ( NM_005531 (del191-393aa/del509-729aa/samesense mut)) lentiviruses were constructed by Genechem Co. Ltd (Shanghai, China).

Techniques: Western Blot, Expressing, Transduction, Mutagenesis, Immunofluorescence, Staining

Journal: Cancer Immunology Research

Article Title: FAM135B Deficiency Inhibits Cytotoxic T-cell Activity in Triple-Negative Breast Cancer by Blocking the IFI16-Dependent STING Pathway

doi: 10.1158/2326-6066.CIR-25-0310

Figure Lengend Snippet: FAM135B interacts with the PYRIN domain of IFI16. A, Bubble plot depicting the functional analysis of the protein list identified by MS. B, The mass spectra of IFI16. C, BT549 and MDA-MB-231 cell lysates were immunoprecipitated using anti-FAM135B or anti-IFI16 antibodies and then were analyzed by immunoblotting. D, 293T cells were co-transfected with Flag-FAM135B and HA-IFI16 plasmids. Cell lysates were immunoprecipitated with either anti-Flag or anti-HA antibodies and then were subjected to immunoblotting analysis using the indicated antibodies. E, BT549 and MDA-MB-231 cells were double-stained with anti-FAM135B and anti-IFI16 antibodies. Confocal microscopy images showed the subcellular localization of FAM135B and IFI16 (scale bar, 10 μm). F, The GST-IFI16 fusion protein was purified and incubated with cell lysates of 293T cells expressing Flag-FAM135B and then analyzed by immunoblotting. Purified GST proteins served as negative controls. G, Flag-tagged full-length FAM135B or truncation mutants were co-transfected with HA-IFI16 into 293T cells. Cell lysates were immunoprecipitated using an anti-Flag antibody and then analyzed by immunoblotting. H, HA-tagged full-length or deletion mutants of IFI16 were co-transfected with Flag-FAM135B into 293T cells. Cell lysates were immunoprecipitated with an anti-HA antibody and were subjected to immunoblotting analysis. FL, full length; IP, immunoprecipitated.

Article Snippet: After blocking with 1% BSA for 1 hour, the cells or sections were incubated overnight at 4°C with the primary antibodies targeting specific proteins: anti-FAM135B (28061-1-AP, Proteintech), anti-IFI16 (sc-8023, Santa Cruz Biotechnology), and anti–IFN regulatory factor 3 (IRF3; 11312-1-AP, Proteintech).

Techniques: Functional Assay, Immunoprecipitation, Western Blot, Transfection, Staining, Confocal Microscopy, Purification, Incubation, Expressing

Journal: Cancer Immunology Research

Article Title: FAM135B Deficiency Inhibits Cytotoxic T-cell Activity in Triple-Negative Breast Cancer by Blocking the IFI16-Dependent STING Pathway

doi: 10.1158/2326-6066.CIR-25-0310

Figure Lengend Snippet: FAM135B competes with TRIM21 for binding to IFI16. A and B, BT549 and MDA-MB-231 cells expressing an empty vector or FAM135B were treated with cycloheximide (CHX; 10 μmol/L) for the indicated time intervals, and cell lysates were subjected to immunoblotting using the indicated antibodies ( A ). The line graph shows the quantification of relative IFI16 levels ( B ). C, TNBC cells expressing an empty vector or FAM135B were treated with MG132 (20 μmol/L) for 6 hours. Cell lysates were immunoprecipitated using an anti-IFI16 antibody and then analyzed by immunoblotting. D, HA-tagged full-length (FL) or deletion mutants of IFI16 were co-transfected with Flag-TRIM21 into 293T cells. Cell lysates were immunoprecipitated using an anti-HA antibody and were subjected to immunoblotting analysis. E, BT549 and MDA-MB-231 cells expressing an empty vector or FAM135B were lysed, immunoprecipitated using an anti-TRIM21 antibody, and then analyzed by immunoblotting. F, Overlapping ubiquitination sites of IFI16 were identified by MS and estimated by the PhosphoSitePlus database. G, BT549 cells expressing an empty vector or TRIM21 were transfected with HA-IFI16 or its mutants (K143R or K561R). Cells were treated with MG132 (20 μmol/L) for 6 hours before harvest, and cell lysates were immunoprecipitated using an anti-HA antibody and analyzed by immunoblotting. H, TNBC cells expressing FAM135B shRNA were transfected with HA-IFI16 or its mutants (K143R or K561R). Cell lysates were analyzed by immunoblotting using the indicated antibodies. I, 293T cells were transfected with Flag-TRIM21, HA-IFI16, and Myc-Ubi or its mutants (K33R or K48R). Cells were treated with MG132 (10 μmol/L) for 6 hours before harvest, and cell lysates were immunoprecipitated using an anti-HA antibody, followed by immunoblotting analysis. IP, immunoprecipitated; p-IRF, phosphorylated IRF; p-STING, phosphorylated STING; p-TBK, phosphorylated TBK; WT, wild type.

Article Snippet: After blocking with 1% BSA for 1 hour, the cells or sections were incubated overnight at 4°C with the primary antibodies targeting specific proteins: anti-FAM135B (28061-1-AP, Proteintech), anti-IFI16 (sc-8023, Santa Cruz Biotechnology), and anti–IFN regulatory factor 3 (IRF3; 11312-1-AP, Proteintech).

Techniques: Binding Assay, Expressing, Plasmid Preparation, Western Blot, Immunoprecipitation, Transfection, Ubiquitin Proteomics, shRNA