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: Representative photomicrographs ( A ) and quantification ( B ) of propidium iodide (PI) staining in sgNC- or sgKO-IFI16-transfected HK-2 cells with H/R treatment ( n = 8). C The extracellular LDH levels in sgNC- or sgKO-IFI16-transfected HK-2 cells with H/R treatment ( n = 6). Representative scatter plots of flow cytometry analysis ( D ) and quantitative data ( E ) depicting the early apoptosis (Annexin V + /7-AAD − , bottom right quadrant, percentages were highlighted in blue) and late apoptosis/necrosis (Annexin V + /7-AAD + , top right quadrant, percentages were highlighted in red) of sgNC- or sgKO-IFI16-transfected HK-2 cells with H/R treatment ( n = 6). F Representative Western blot gel documents and summarized data showing the protein levels of p-p53 in sgNC- or sKO-IFI16-transfected HK-2 cells with H/R treatment ( n = 6). G Representative Western blot gel documents and summarized data showing the protein levels of p53 and BAX and caspase-3 cleavage in sgNC- or sgKO-IFI16-transfected HK-2 cells with H/R treatment ( n = 3). Representative photomicrographs ( H ) and quantification ( I ) of terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick-end labeling (TUNEL) assays of the kidneys from different groups of IRI model mice to assess renal cell death ( n = 8). Nuclei were revealed by using DAPI staining. Data are represented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For inhibitor treatment, LV-IFI16-GFP HK-2 cells were pre-incubated with PJ34 (50 μM, MedChemExpress, HY-13688A), Ku55933 (10 μM, MedChemExpress, HY-12016), Z-VAD-FMK (20 μM, MedChemExpress, HY-16658B), Nec-1S (10 μM, MedChemExpress, HY-14622A), Ferrostatin-1 (10 μM, MedChemExpress, HY-100579) for 6 h. Subsequently, the cells were subjected to hypoxia with continuous inhibitor supplementation, followed by reoxygenation for 2 or 24 h.

Techniques: Staining, Transfection, Flow Cytometry, Western Blot, End Labeling, TUNEL Assay

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 Representative photomicrographs and quantification of 4HNE immunohistochemistry in the kidneys from different groups of IRI model mice ( n = 8). B Representative photomicrographs and quantification of MDA immunohistochemistry in the kidneys from different groups of IRI model mice ( n = 8). C Quantitative analysis of MDA levels in sgNC- or sgKO-IFI16-transfected HK-2 cells with H/R treatment ( n = 6). Representative photomicrographs ( D ) and quantification ( E ) of BODIPY-C11 probe staining in sgNC- or sgKO-IFI16-transfected HK-2 cells with H/R treatment ( n = 8). Representative photomicrographs ( F ) and quantification ( G ) of ACSL4 immunohistochemistry in the kidneys from different groups of IRI model mice ( n = 8). Representative Western blot gel documents ( H ) and summarized data ( I ) showing the protein levels of ACSL4 in the cortex of kidneys from different groups of IRI model mice ( n = 6). J Representative Western blot gel documents and summarized data showing the protein levels of ACSL4, GPX4, and SLC7A11 in sgNC- or sgKO-IFI16-transfected HK-2 cells with H/R treatment ( n = 6). K Quantitative analysis of GSH/GSSG levels in sgNC- or sgKO-IFI16-transfected HK-2 cells with H/R treatment ( n = 6). Data are represented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For inhibitor treatment, LV-IFI16-GFP HK-2 cells were pre-incubated with PJ34 (50 μM, MedChemExpress, HY-13688A), Ku55933 (10 μM, MedChemExpress, HY-12016), Z-VAD-FMK (20 μM, MedChemExpress, HY-16658B), Nec-1S (10 μM, MedChemExpress, HY-14622A), Ferrostatin-1 (10 μM, MedChemExpress, HY-100579) for 6 h. Subsequently, the cells were subjected to hypoxia with continuous inhibitor supplementation, followed by reoxygenation for 2 or 24 h.

Techniques: Immunohistochemistry, Transfection, Staining, Western Blot

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: Quantitative analysis of iron ( A ) and Fe 2+ ( B ) levels in sgNC- or sgKO-IFI16-transfected HK-2 cells with H/R treatment ( n = 6). Representative photomicrographs ( C ) and quantification ( D ) of FerroOrange staining in sgNC- or sgKO-IFI16-transfected HK-2 cells with H/R treatment ( n = 8). FerroOrange (Orange) exhibited the localization of intracellular free iron in living cells. E Representative Western blot gel documents and summarized data showing the protein levels of FTH1 and FTL in sgNC- or sgKO-IFI16-transfected HK-2 cells with H/R treatment ( n = 6). F The subcellular localization of MTF1 in sgNC- or sgKO-IFI16-transfected HK-2 cells with H/R treatment ( n = 8). Representative photomicrographs are shown to visualize the subcellular locations of MTF1 upon different groups. G Representative western blot gel documents and summarized data showing the protein levels of FTH1 and FTL in the cortex of kidneys from different groups of IRI model mice ( n = 6). Data are represented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For inhibitor treatment, LV-IFI16-GFP HK-2 cells were pre-incubated with PJ34 (50 μM, MedChemExpress, HY-13688A), Ku55933 (10 μM, MedChemExpress, HY-12016), Z-VAD-FMK (20 μM, MedChemExpress, HY-16658B), Nec-1S (10 μM, MedChemExpress, HY-14622A), Ferrostatin-1 (10 μM, MedChemExpress, HY-100579) for 6 h. Subsequently, the cells were subjected to hypoxia with continuous inhibitor supplementation, followed by reoxygenation for 2 or 24 h.

Techniques: Transfection, Staining, Western Blot

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 Representative Western blot gel documents and summarized data showing the protein levels of p-ATM, p-KAP1 and p-NBS1 in sgNC- or sgKO-IFI16-transfected HK-2 cells with H/R 2 h treatment ( n = 6). B Representative photomicrographs and quantification of γ-H2AX immunofluorescence staining in sgNC- or sgKO-IFI16-transfected HK-2 cells with H/R treatment ( n = 8). C Representative Western blot gel documents and summarized data showing the protein levels of p-ATM in the cortex of kidneys from different groups of IRI model mice ( n = 6). D Representative Western blot gel documents and summarized data showing the protein levels of GPX4, SLC7A11, FTH1 and FTL in LV-IFI16-GFP-transfected HK-2 cells with H/R 24 h treatment with or without Ku-55933 (10 µM) pretreatment ( n = 3). E Representative photomicrographs and quantification of FerroOrange staining in LV-IFI16-GFP-transfected HK-2 cells with H/R 24 h treatment with or without Ku-55933 pretreatment ( n = 8). F Quantitative analysis of MDA levels in LV-IFI16-GFP-transfected HK-2 cells with H/R 24 h treatment with or without Ku-55933 pretreatment ( n = 6). G Representative photomicrographs and quantification of propidium iodide (PI) staining in LV-IFI16-GFP-transfected HK-2 cells with H/R 24 h treatment with or without Ku-55933 pretreatment ( n = 8). H The extracellular LDH levels in LV-IFI16-GFP-transfected HK-2 cells with H/R 24 h treatment and with or without Ku-55933 pretreatment ( n = 6). Data are represented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For inhibitor treatment, LV-IFI16-GFP HK-2 cells were pre-incubated with PJ34 (50 μM, MedChemExpress, HY-13688A), Ku55933 (10 μM, MedChemExpress, HY-12016), Z-VAD-FMK (20 μM, MedChemExpress, HY-16658B), Nec-1S (10 μM, MedChemExpress, HY-14622A), Ferrostatin-1 (10 μM, MedChemExpress, HY-100579) for 6 h. Subsequently, the cells were subjected to hypoxia with continuous inhibitor supplementation, followed by reoxygenation for 2 or 24 h.

Techniques: Western Blot, Transfection, Immunofluorescence, Staining

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 Representative Western blot gel documents of the co-immunoprecipitation (co-IP) assay detecting the interaction between IFI16 and PARP1 in HK-2 cells overexpressing IFI16-GFP with H/R 2 h treatment. SE short exposure, LE long exposure. B Representative Western blot gel documents and summarized data showing the levels of protein PARylation in sgNC- or sgKO-IFI16-transfected HK-2 cells with H/R 24 h treatment ( n = 3). C Quantitative analysis of NAD + /NADH levels in sgNC- or sgKO-IFI16-transfected HK-2 cells with H/R 24 h treatment ( n = 6). D Quantitative analysis of NAD + /NADH levels in LV-IFI16-GFP-transfected HK-2 cells with H/R 24 h treatment with or without PJ34 (10 µM) pretreatment ( n = 6). E The extracellular LDH levels in LV-IFI16-GFP-transfected HK-2 cells with H/R 24 h treatment with or without PJ34 pretreatment ( n = 6). Representative photomicrographs ( F ) and quantification ( G ) of propidium iodide (PI) staining in LV-IFI16-GFP-transfected HK-2 cells with H/R 24 h treatment with or without PJ34 pretreatment ( n = 8). Representative photomicrographs ( H ) and quantification ( I ) of FerroOrange staining in LV-IFI16-GFP-transfected HK-2 cells with H/R 24 h treatment with or without PJ34 pretreatment ( n = 8). J Quantitative analysis of MDA levels in LV-IFI16-GFP-transfected HK-2 cells with H/R 24 h treatment with or without PJ34 pretreatment ( n = 6). K Representative Western blot gel documents and summarized data showing the protein levels of GPX4, SLC7A11, FTH1 and FTL in LV-IFI16-GFP-transfected HK-2 cells with H/R 24 h treatment with or without PJ34 pretreatment ( n = 3). Data are represented as the mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: For inhibitor treatment, LV-IFI16-GFP HK-2 cells were pre-incubated with PJ34 (50 μM, MedChemExpress, HY-13688A), Ku55933 (10 μM, MedChemExpress, HY-12016), Z-VAD-FMK (20 μM, MedChemExpress, HY-16658B), Nec-1S (10 μM, MedChemExpress, HY-14622A), Ferrostatin-1 (10 μM, MedChemExpress, HY-100579) for 6 h. Subsequently, the cells were subjected to hypoxia with continuous inhibitor supplementation, followed by reoxygenation for 2 or 24 h.

Techniques: Western Blot, Co-Immunoprecipitation Assay, Transfection, Staining

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: For inhibitor treatment, LV-IFI16-GFP HK-2 cells were pre-incubated with PJ34 (50 μM, MedChemExpress, HY-13688A), Ku55933 (10 μM, MedChemExpress, HY-12016), Z-VAD-FMK (20 μM, MedChemExpress, HY-16658B), Nec-1S (10 μM, MedChemExpress, HY-14622A), Ferrostatin-1 (10 μM, MedChemExpress, HY-100579) for 6 h. Subsequently, the cells were subjected to hypoxia with continuous inhibitor supplementation, followed by reoxygenation for 2 or 24 h.

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

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