Journal: Translational Oncology

Article Title: IL1RA inhibits the progression of oral squamous cell carcinoma by mediating type Ⅰ interferon response

doi: 10.1016/j.tranon.2025.102428

Figure Lengend Snippet: IL1RA is associated with oncogenic signaling pathways and type I interferon response in OSCC in vitro . A. Volcano plots visualizing 132 DEGs between CAL27 cells transfected with NC and oeIL1RA. Down-regulated, up-regulated and non-regulated genes were labeled in blue, red, and grey colors, respectively. B. GSEA showed less enriched cancer-related features or processes in CAL27 cells overexpressing IL1RA. C. GSEA showed that IL1RA overexpression was significantly associated with the type I interferon response. D. A Venn diagram visualizing 45 (61.6 %) OSCC patients simultaneously carrying mutations in the IL1RA, IFNA , and IFNB genes in the TCGA dataset.

Article Snippet: Slices were then incubated with diluted primary antibodies against IL1RA (NBP1-32568, Novusbio, 1:600), E-Cadherin (#13116, CST, 1:400), N-Cadherin (#3195, CST, 1:100), Vimentin (#5741, CST, 1:100), IFNA (18013-AP, Proteintech, 1:400), and IFNB (27506-AP, Proteintech, 1:400) overnight at 4° C, and the secondary antibody (horseradish peroxidase-labeled goat anti-rabbit) at room temperature for 2 h. Cell nuclei were dyed with a DAB staining solution.

Techniques: Protein-Protein interactions, In Vitro, Transfection, Labeling, Over Expression

Journal: Translational Oncology

Article Title: IL1RA inhibits the progression of oral squamous cell carcinoma by mediating type Ⅰ interferon response

doi: 10.1016/j.tranon.2025.102428

Figure Lengend Snippet: Overexpression of IL1RA up-regulates type I interferon proteins in OSCC in vitro . A. H&E (the first lane) and IHC staining (the latter three lanes) of IFNA and IFNB in OSCC specimens of the high and low IL1RA expression groups (n=30, scale bar=100 μm). B-C. The mRNA (B) and protein expressions (C) of IFNA and IFNB in OSCC specimens of the high and low IL1RA expression groups. * P <0.05, and ** P <0.01 vs. NC group.

Article Snippet: Slices were then incubated with diluted primary antibodies against IL1RA (NBP1-32568, Novusbio, 1:600), E-Cadherin (#13116, CST, 1:400), N-Cadherin (#3195, CST, 1:100), Vimentin (#5741, CST, 1:100), IFNA (18013-AP, Proteintech, 1:400), and IFNB (27506-AP, Proteintech, 1:400) overnight at 4° C, and the secondary antibody (horseradish peroxidase-labeled goat anti-rabbit) at room temperature for 2 h. Cell nuclei were dyed with a DAB staining solution.

Techniques: Over Expression, In Vitro, Immunohistochemistry, Expressing

Journal: Translational Oncology

Article Title: IL1RA inhibits the progression of oral squamous cell carcinoma by mediating type Ⅰ interferon response

doi: 10.1016/j.tranon.2025.102428

Figure Lengend Snippet: IL1RA promotes the expressions of IFNA and IFNB in the OSCC xenografts and their release in OSCC cells. A. IHC staining of positive expressions of IFNA and IFNB in OSCC xenografts of oeIL1RA group and NC group in vivo (scale bar=100 μm). B. The mRNA levels of IFNA and IFNB in OSCC xenografts of the oeIL1RA group and NC group in vivo . C. ELISA showed contents of IFNA and IFNB in the cell supernatant of the oeIL1RA group and NC group in vitro . * P <0.05, and ** P <0.01 vs. NC group.

Article Snippet: Slices were then incubated with diluted primary antibodies against IL1RA (NBP1-32568, Novusbio, 1:600), E-Cadherin (#13116, CST, 1:400), N-Cadherin (#3195, CST, 1:100), Vimentin (#5741, CST, 1:100), IFNA (18013-AP, Proteintech, 1:400), and IFNB (27506-AP, Proteintech, 1:400) overnight at 4° C, and the secondary antibody (horseradish peroxidase-labeled goat anti-rabbit) at room temperature for 2 h. Cell nuclei were dyed with a DAB staining solution.

Techniques: Immunohistochemistry, In Vivo, Enzyme-linked Immunosorbent Assay, In Vitro

Journal: Biochemical Journal

Article Title: Suppression of interferon β gene transcription by inhibitors of bromodomain and extra-terminal (BET) family members

doi: 10.1042/BJ20141523

Figure Lengend Snippet: RAW cells were incubated for 1 h without (−) or with (+) 1.0 μM JQ1 or 1.0 μM BI2536 and then stimulated for 8 h without (−) or with (+) poly(I:C) (10 μg/ml) ( A and C ) or for 4 h without (−) or with (+) LPS (100 ng/ml) ( B and D ). At each time point, the total RNA was extracted from the cells and Ifnb mRNA was quantified by qPCR ( A and B ) and the concentration of IFNβ in the cell culture medium was determined by ELISA ( C and D ). Results are means+S.E.M for triplicate determinations. ( A ) and ( B ) show the fold increase in mRNA levels relative to the values measured in cells that had not been stimulated with LPS or poly(I:C).

Article Snippet: The level of secreted IFNβ in the cell culture medium was determined using the Verikine mouse and human IFNβ ELISA kits (PBL Interferon Source) or the LEGEND MAXTM Mouse IFN-β ELISA Kit (BioLegend) following the manufacturer's protocol.

Techniques: Incubation, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Biochemical Journal

Article Title: Suppression of interferon β gene transcription by inhibitors of bromodomain and extra-terminal (BET) family members

doi: 10.1042/BJ20141523

Figure Lengend Snippet: ( A and B ) Gen2.2 cells were incubated for 1 h with or without BI-2536 (1.0 μM), JQ1 (1.0 μM) or I-BET151 (1.0 μM) and then stimulated for 8 h with CL097 (1.0 μg/ml) ( A ) or for 12 h with ODN1826 (1.0 μM) ( B ). The concentration of IFNβ in the culture medium was measured by ELISA. Results are means+S.D. from two independent experiments each performed in duplicate. ( C ) Gen2.2 cells were incubated for 1 h with or without BI-2536 (1.0 μM), JQ1 (1.0 μM), I-BET151 (1.0 μM) or BI-605906 (5.0 μM) and then stimulated for 1 h with CL097. Staining with anti-IRF5, or DAPI to reveal nuclei, followed by deconvolution microscopy was performed as described in the Materials and methods section. ( D ) Gen2.2 cells were incubated for 1 h with BI-2536 (1 μM) or JQ1 (1 μM), then stimulated for 1 h with the TLR7 agonist CL097 (1 μg/ml), cross-linked and lysed. Chromatin was sheared by sonication and ChIP was performed using anti-IRF5. The enrichment of the Ifnb promoter was measured by qPCR, normalizing to input. Results are means+S.D. similar results were obtained in three independent experiments each performed in duplicate. IP, immunoprecipitation.

Article Snippet: The level of secreted IFNβ in the cell culture medium was determined using the Verikine mouse and human IFNβ ELISA kits (PBL Interferon Source) or the LEGEND MAXTM Mouse IFN-β ELISA Kit (BioLegend) following the manufacturer's protocol.

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Microscopy, Sonication, Immunoprecipitation

Journal: Emerging microbes & infections

Article Title: Novel transcription and replication-competent virus-like particles system modelling the Nipah virus life cycle.

doi: 10.1080/22221751.2024.2368217

Figure Lengend Snippet: Figure 6. Utility of trVLP-NiV in evaluating the effects of different kinds of antiviral agents. (A) 293T-NPL cells were infected with trVLP-NiV and cultured with 10 μM of FEMA 4809, remdesivir, and ribavirin, respectively. DMSO was used as a control. After 24 h infection, RLuc activity was quantified and normalized to DMSO-treated cells. Antiviral effect of ribavirin (B), remdesivir (C), IFN-α (D), and IFN-β (E). The 293T-NPL cells were infected with trVLP-NiV and treated with indicated concentrations of ribavirin or remdesivir. For IFN-α and IFN-β, cells were pre-treated with IFN-α or IFN-β for 8 h before infection. After 24 h of infection, RLuc activity (dark orange) and cell viability (blue) were measured. (F to H) Evaluating neutralizing activity with trVLP-NiV. The indicated concentrations of mAbs were incubated with trVLP-NiV for 1 h at 37 °C. In addition, residual infectivity was determined using 293T-NPL cells. IC50, the half-maximum inhibitory concentration; CC50, 50% cytotoxic concentration. N = 3. Error bars indi cate SEM. NS, no significance; *P < 0.05; **P < 0.01. Significance assessed by student’s t-test.

Article Snippet: For interferon-α (IFN-α) and IFN-β treatment, the cells were pre-treated with different concentrations of IFN-α (MCE, HYP73246) and IFN-β (Sino Biological, 10704-HNAS-5) 8 h prior to infection.

Techniques: Infection, Cell Culture, Control, Activity Assay, Incubation, Concentration Assay

Journal: EMBO Reports

Article Title: RNF144B negatively regulates antiviral immunity by targeting MDA5 for autophagic degradation

doi: 10.1038/s44319-024-00256-w

Figure Lengend Snippet: ( A ) Rnf144b +/+ and Rnf144b −/− MEFs were infected with EMCV (MOI = 1) for indicated time points, and then cell lysates were analyzed for EMCV replication level by qPCR. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student’s t-test. **** P = 0.000039 (4 h), *** P = 0.00050 (8 h), **** P = 0.000040 (12 h). ( B ) Rnf144b +/+ and Rnf144b −/− MEFs were infected with EMCV (MOI = 1) for indicated time points. The cell supernatant was harvested and analyzed by TCID 50 assay. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student’s t-test. ** P = 0.00540 (8 h), ** P = 0.00820 (12 h). ( C ) Western blot analysis of the indicated signaling proteins in MEFs from Rnf144b +/+ or Rnf144b −/− mice infected with EMCV (MOI = 1) for the indicated time periods. ( D ) Rnf144b +/+ and Rnf144b −/− MEFs were infected with EMCV (MOI = 1). After 12 h, cell lysates were analyzed for Ifnb1, TNF-α, IL-6 mRNA level by qPCR. n = 3 biological replicates. Statistical significance was determined by two-tailed unpaired Student’s t-test. **** P = 0.000009 (Ifnb1), *** P = 0.00090 (TNF-α), *** P = 0.00030 (IL-6). ( E ) Rnf144b +/+ and Rnf144b −/− MEFs were infected with EMCV (MOI = 1). 12 h post-infection, cells were treated by CHX (30 μM) for indicated time points. Protein extracts were used for immunoblot analysis of the endogenous MDA5 protein level. ( F , G ) Rnf144b +/+ and Rnf144b −/ − mice were intraperitoneally injected EMCV (5 × 10 6 PFU) for 48 h ( n = 5 per group). qPCR analysis of EMCV replication level in heart and brain, and mRNA level of Ifnb1, TNF-α, IL-6 in brain ( G ). Statistical significance was determined by two-tailed unpaired Student’s t-test. ** P = 0.00530 (heart), * P = 0.02620 (brain), *** P = 0.00030 (Ifnb1), ** P = 0.00170 (TNF-α), *** P = 0.00030 (IL-6). ( H ) ELISA of IFN-β production in serum of Rnf144b +/+ and Rnf144b −/− mice that were intraperitoneally injected with EMCV (5 × 10 6 PFU) for 12 h ( n = 3 per group). Statistical significance was determined by two-tailed unpaired Student’s t-test. **** P = 0.000020. ( I ) Survival of Rnf144b +/+ and Rnf144b −/− mice ( n = 20 per group) intraperitoneally infected with EMCV (6 × 10 7 PFU). Significance was tested using log-rank (Mantel-Cox) test, *** P = 0.00030. Data information: Data shown are representative of at least three biological replicates, with each data point representing a biological experiment. Error bars are presented as mean ± SD. Statistical significance was determined by Student’s t-test. .

Article Snippet: Mouse Interferon β (IFN-β/IFNB) ELISA Kit , CUSABIO , Cat#E04945m.

Techniques: Infection, Two Tailed Test, Western Blot, Injection, Enzyme-linked Immunosorbent Assay

Journal: EMBO Reports

Article Title: RNF144B negatively regulates antiviral immunity by targeting MDA5 for autophagic degradation

doi: 10.1038/s44319-024-00256-w

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Mouse Interferon β (IFN-β/IFNB) ELISA Kit , CUSABIO , Cat#E04945m.

Techniques: Recombinant, Sequencing, shRNA, Mutagenesis, RNA Extraction, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal: International immunopharmacology

Article Title: HERC5 induces podocyte injury in LN by mediated IRF3 ISGylation to promote the production and overactivation of IFN-β in podocytes.

doi: 10.1016/j.intimp.2025.115059

Figure Lengend Snippet: Fig. 3. Both LN plasma and IFN-β stimulations induce podocyte injury through excessive IFN-I pathway activation. A-D. HPCs were treated with LN plasma for 24 h and 48 h. Representative western blot images and quantitation of Podocin, WT1 and HERC5. E. Real time qPCR showing mRNA levels of Ifn-b1, Tnf-α, Il-6, Cxcl10 and Il-10 treated with LN plasma for 48 h. F-G. HPCs were treated with IFN-β1(IFN-β, 1 ng/ml) for 24 h. Representative western blot images and quantitation of Podocin and WT1. H-I. Representative western blot images and quantitation of HERC5 treated with IFN-β for 15 min, 30 min, 60 min, 120 min, 240 min. J. Real time qPCR showing mRNA levels of Ifn-b1, Tnf-α, Il-6, Cxcl10 and Il-10 treated with IFN-β for 30 min. GAPDH was used for normalization. n = 3 independent experiments. Normal distributions were tested by Shapiro–Wilk method. P values were determined by Student’s t-test for 2 groups or one-way ANOVA followed by the LSD test for more than 2 groups and data were presented as mean ± SD of normally distributions data. Otherwise, P values were determined by Mann-Whitney U test for 2 groups and data were presented as median (interquartile range) of not normally distributed data.

Article Snippet: Different treatments of the cells were separately represented as follows: (1) The HPCs were serum-starved overnight and exposed to LN plasma (10 % in RMPI-1640) for 0, 24, 48 h. (2) The HPCs were treated with IFN-β (1 ng/ml; MCE, HY-P73128, Shanghai, China) for 0, 15, 30, 60, 120, 240 or 0, 24 h. (3) The HPCs were transfected with siRNA targeting HERC5 (siRNA-HERC5) or negative control (siRNA-NC) followed with LN plasma or IFN-β stimulation. (4) The HPCs were transfected with vector overexpressing HERC5 (pcDNA3.1- myc-his-herc5) or negative control (pcDNA3.1). (5) The HPCs were co-transfected with vector overexpressing HERC5 (pcDNA3.1-myc-his-herc5) and siRNA targeting IRF3 (siRNA-IRF3) or negative control (siRNA-NC) or co-transfected with negative control (pcDNA3.1) and siRNA targeting IRF3 (siRNA-IRF3) or negative control (siRNA-NC).

Techniques: Clinical Proteomics, Activation Assay, Western Blot, Quantitation Assay, MANN-WHITNEY

Journal: International immunopharmacology

Article Title: HERC5 induces podocyte injury in LN by mediated IRF3 ISGylation to promote the production and overactivation of IFN-β in podocytes.

doi: 10.1016/j.intimp.2025.115059

Figure Lengend Snippet: Fig. 6. HERC5 positively regulates IRF3 phosphorylation and nuclear translocation A-D. Representative western blot images and quantitation of p-IRF3 (Ser385), p- IRF3 (Ser396) and total IRF3 levels treated with IFN-β for 15 min, 30 min, 60 min, 120 min, 240 min. E-F. Representative western blot images and quantitation of HERC5, p-IRF3 (Ser385), p-IRF3 (Ser396) and total IRF3 levels in nuclear and supernatant of HPCs treated with IFN-β for 30 min. G. The localization and expression of p-IRF3 (Ser385) and p-IRF3 (Ser396) in HPCs was detected by IF treated with IFN-β for 30 min. H-I. Representative western blot images and quantitation of HERC5, p-IRF3 (Ser385), p-IRF3 (Ser396) and total IRF3 levels in HPCs pretreated with NC and siHERC5 and treated with IFN-β for 30 min. J-K. Representative western blot images and quantitation of HERC5, p-IRF3 (Ser385), p-IRF3 (Ser396) and total IRF3 levels in nuclear and supernatant of HPCs pretreated with NC and siHERC5 and treated with IFN-β for 30 min. L-M. Representative western blot images and quantitation of HERC5, p-IRF3 (Ser385), p-IRF3 (Ser396) and total IRF3 levels in HPCs transfected with pcDNA3.1 and Myc-his-HERC5. N-O. Representative western blot images and quantitation of HERC5, p-IRF3 (Ser385), p-IRF3 (Ser396) and total IRF3 levels in nuclear and supernatant of HPCs transfected with pcDNA3.1 and Myc-his-HERC5. n = 3 independent experiments. Data were presented as mean ± SD and normal distributions were tested by Shapiro–Wilk method, which showed that all the data were normally distributed. Student’s t-test was used for 2 groups and one-way ANOVA followed by the LSD test for more than 2 groups and data were presented as mean ± SD.

Article Snippet: Different treatments of the cells were separately represented as follows: (1) The HPCs were serum-starved overnight and exposed to LN plasma (10 % in RMPI-1640) for 0, 24, 48 h. (2) The HPCs were treated with IFN-β (1 ng/ml; MCE, HY-P73128, Shanghai, China) for 0, 15, 30, 60, 120, 240 or 0, 24 h. (3) The HPCs were transfected with siRNA targeting HERC5 (siRNA-HERC5) or negative control (siRNA-NC) followed with LN plasma or IFN-β stimulation. (4) The HPCs were transfected with vector overexpressing HERC5 (pcDNA3.1- myc-his-herc5) or negative control (pcDNA3.1). (5) The HPCs were co-transfected with vector overexpressing HERC5 (pcDNA3.1-myc-his-herc5) and siRNA targeting IRF3 (siRNA-IRF3) or negative control (siRNA-NC) or co-transfected with negative control (pcDNA3.1) and siRNA targeting IRF3 (siRNA-IRF3) or negative control (siRNA-NC).

Techniques: Phospho-proteomics, Translocation Assay, Western Blot, Quantitation Assay, Expressing, Transfection

Journal: International immunopharmacology

Article Title: HERC5 induces podocyte injury in LN by mediated IRF3 ISGylation to promote the production and overactivation of IFN-β in podocytes.

doi: 10.1016/j.intimp.2025.115059

Figure Lengend Snippet: Fig. 7. HERC5 inhibited ubiquitination of IRF3 by mediating its ISGylation. Co-Immunoprecipitation (Co-IP) demonstrated that endogenous IRF3 bound to HERC5 and ISG15, in the meantime, IFN-β stimulation (1 ng/ml for 30 min) increased the interaction and the ISGylation (ISG15) of IRF3. B. Co-IP demonstrated that overexpression of HERC5 increased the ISGylation of IRF3 by transfected with pcDNA3.1-Myc-his-HERC5 (Myc-his-HERC5) than control (pcDNA3.1). C. Co-IP demonstrated that HERC5 knockdown decreased the ISGylation of IRF3 by transfected with siRNA. D. Co-IP demonstrated that HERC5 knockdown increased the ubiquitination level of IRF3. E. Co-IP analysis demonstrated HERC5 enhances IRF3 ISGylation, thereby inhibiting its ubiquitination and degradation via proteasomes, transfected with pcDNA3.1, pcDNA3.1-Myc-ISG15(Myc-ISG15) or pcDNA3.1-Myc-his-HERC5 (Myc-his-HERC5) treated by MG132(15 μM) for 8 h or not, under IFN- β(1 ng/ml) for 30 min stimulation. F-G. Western blot demonstrated that overexpression HERC5 influence the IRF3 protein stability and prolongs its half-life treated HPCs with cycloheximide (CHX) (100 μg/ml).

Article Snippet: Different treatments of the cells were separately represented as follows: (1) The HPCs were serum-starved overnight and exposed to LN plasma (10 % in RMPI-1640) for 0, 24, 48 h. (2) The HPCs were treated with IFN-β (1 ng/ml; MCE, HY-P73128, Shanghai, China) for 0, 15, 30, 60, 120, 240 or 0, 24 h. (3) The HPCs were transfected with siRNA targeting HERC5 (siRNA-HERC5) or negative control (siRNA-NC) followed with LN plasma or IFN-β stimulation. (4) The HPCs were transfected with vector overexpressing HERC5 (pcDNA3.1- myc-his-herc5) or negative control (pcDNA3.1). (5) The HPCs were co-transfected with vector overexpressing HERC5 (pcDNA3.1-myc-his-herc5) and siRNA targeting IRF3 (siRNA-IRF3) or negative control (siRNA-NC) or co-transfected with negative control (pcDNA3.1) and siRNA targeting IRF3 (siRNA-IRF3) or negative control (siRNA-NC).

Techniques: Ubiquitin Proteomics, Immunoprecipitation, Co-Immunoprecipitation Assay, Over Expression, Transfection, Control, Knockdown, Western Blot

Journal: International immunopharmacology

Article Title: HERC5 induces podocyte injury in LN by mediated IRF3 ISGylation to promote the production and overactivation of IFN-β in podocytes.

doi: 10.1016/j.intimp.2025.115059

Figure Lengend Snippet: Fig. 8. Knockdown of IRF3 can alleviate the overactivation of IFN-β induced by HERC5 overexpression, thereby reducing podocyte damage. Real time qPCR showing mRNA levels of Ifn-b1, Tnf-α, Il-6, Cxcl10 and Il-10 in HPCs transfected with NC or siIRF3 (siRNA-IRF3) after transfected with Myc-his-HERC5 or pcDNA3.1 for 24 h. B-C. Representative western blot images and quantitation of IRF3, Podocin and WT1 levels in HPCs treated as A. n = 3 independent experiments. Normal distri butions were tested by Shapiro–Wilk method. P values were determined by Student’s t-test or Mann-Whitney U test. Data were presented as mean ± SD or median (interquartile range).

Article Snippet: Different treatments of the cells were separately represented as follows: (1) The HPCs were serum-starved overnight and exposed to LN plasma (10 % in RMPI-1640) for 0, 24, 48 h. (2) The HPCs were treated with IFN-β (1 ng/ml; MCE, HY-P73128, Shanghai, China) for 0, 15, 30, 60, 120, 240 or 0, 24 h. (3) The HPCs were transfected with siRNA targeting HERC5 (siRNA-HERC5) or negative control (siRNA-NC) followed with LN plasma or IFN-β stimulation. (4) The HPCs were transfected with vector overexpressing HERC5 (pcDNA3.1- myc-his-herc5) or negative control (pcDNA3.1). (5) The HPCs were co-transfected with vector overexpressing HERC5 (pcDNA3.1-myc-his-herc5) and siRNA targeting IRF3 (siRNA-IRF3) or negative control (siRNA-NC) or co-transfected with negative control (pcDNA3.1) and siRNA targeting IRF3 (siRNA-IRF3) or negative control (siRNA-NC).

Techniques: Knockdown, Over Expression, Transfection, Western Blot, Quantitation Assay, MANN-WHITNEY

Journal: Nature medicine

Article Title: cGAS drives noncanonical-inflammasome activation in age-related macular degeneration.

doi: 10.1038/nm.4450

Figure Lengend Snippet: Figure 1 Caspase-4/11 in geographic atrophy and RPE degeneration. (a) Immunoblots for pro-caspase-4 (pro-Casp4) and the p30 cleavage product of caspase-4 (Casp4 p30) in the RPE in human eyes with geographic atrophy (dry AMD) and in unaffected controls (ctr). Molecular-weight markers (kDa) are indicated to the left of the blots. The bar graph shows densitometry of the bands corresponding to caspase-4 p30 in the control (normal) and dry AMD samples, normalized to the loading control (AU, arbitrary units). Data are mean ± s.d.; n = 3 control eyes; n = 6 dry AMD eyes; *P = 0.002, two-tailed t test. (b) Immunoblots for pro-Casp4 and Casp4 p30 in human RPE cells transfected with Alu RNA or mock transfected (mock; transfection mixture without Alu RNA); transfected with Alu expression plasmid (pAlu) or empty vector (pNull); or transfected with DICER1 or control antisense oligonucleotides (AS). Avg, average. (c) Immunoblot for pro-caspase-11 (pro-Casp11) and the p30 cleavage product of caspase-11 (Casp11 p30) in RPE tissue of WT mice injected subretinally with Alu RNA or vehicle (vehi). p43/37 denotes p43 and/or p37. n = 3 mice per group. (d,e) Top, fundus photographs of the retinas of WT (n = 8 eyes) and Casp11−/− (n = 10 eyes) mice (d) and Casp11−/− mice expressing a human caspase-4 transgene (Casp11−/−; hCasp4Tg) (n = 8 eyes) (e) injected with vehicle (n = 12 eyes) or Alu RNA (n = 14 eyes). Bottom, immunostaining with antibody to zonula occludens-1 (ZO-1) to visualize RPE cellular boundaries. (f) Immunoblots of pro-caspase-1 (pro-Casp1) and the p20 cleavage product of caspase-1 (Casp1 p20) in RPE tissue of WT and Casp11−/− mice injected subretinally with vehicle or Alu RNA. n = 3 mice per group. (g) Immunoblots of pro-caspase-1 and the p20 cleavage product of caspase-1 in WT and Casp11−/− mouse RPE cells treated with Alu RNA. (h) IL-18 secretion by WT and Casp11−/− mouse RPE cells mock transfected or transfected with Alu RNA. n = 3 independent experiments. Data presented are mean ± s.d.; *P = 0.014, two-tailed t test. (i) Top, fundus photographs of the retinas of WT mice (n = 8 eyes), mice deficient in caspase-1 and caspase-11 (Casp1−/−; Casp11129mt/129mt) (n = 7 eyes), or Casp1−/−; Casp11129mt/129mt mice expressing functional mouse caspase-11 from a bacterial artificial chromosome transgene (Casp1−/−; Casp11129mt/129mt; Casp11Tg) (n = 8 eyes) subretinally injected with vehicle (n = 11 eyes) or Alu RNA (n = 12 eyes). Bottom, immunostaining with antibody to ZO-1. For all immunoblots, cropped gel images of bands of interest of representative immunoblots from three independent experiments and densitometric analysis (mean (s.e.m.)) are shown. Tubulin, β-actin, or vinculin was used as a loading control, as indicated. In d, e, and i, the degenerated retinal area is outlined by blue arrowheads in the fundus images; loss of regular hexagonal cellular boundaries in ZO-1-stained flat mounts is indicative of degenerated RPE. RPE degeneration was assessed by binary quantification (healthy (%)) and morphometric quantification (PM, polymegethism (mean (s.e.m.))) (Fisher’s exact test for binary; two-tailed t test for morphometry; *P < 0.05; **P < 0.01; ***P < 0.001). For all micrographs, scale bars, 20 µm.

Article Snippet: In vivo transfection of plasmids expressing Alu sequences (pAlu)65,66, emptycontrol vector (pNull), Flag-cGAS (pFlag-cGAS), Flag-GFP, mouse mature IL-18 (pIL-18ss)57,67, WT mouse gasdermin D (pGSDMD-WT), the p30-cleavageincompetent mutant mouse gasdermin D (pGSMDD-D276A)19, IFN-β (Origene, MR226101), or mtDNA (10 ng) was achieved with 10% Neuroporter (Genlantis) as previously described4,5.

Techniques: Western Blot, Molecular Weight, Control, Two Tailed Test, Transfection, Expressing, Plasmid Preparation, Injection, Immunostaining, Functional Assay, Staining

Journal: Nature medicine

Article Title: cGAS drives noncanonical-inflammasome activation in age-related macular degeneration.

doi: 10.1038/nm.4450

Figure Lengend Snippet: Figure 2 Gasdermin D in geographic atrophy and RPE degeneration. (a) Top, fundus photographs of eyes of WT (n = 6 eyes) and Gsdmd−/− (n = 10 eyes) mice subretinally injected with vehicle (n = 7 eyes) or Alu RNA (n = 9 eyes). Bottom, immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of the same eyes showing RPE cell boundaries. (b) IL-18 secretion by WT and Gsdmd−/− mouse RPE cells mock transfected or transfected with Alu RNA. n = 3 independent experiments. Data are mean ± s.d.; *P = 0.01, two-tailed t test. (c) Immunoblots of pro-caspase-1 (pro-Casp1) and the p10 cleavage product of caspase-1 (Casp1 p10) in WT and Gsdmd−/− mouse RPE cells mock transfected or transfected with Alu RNA. (d) Immunoblots of pro-caspase-11 (pro-Casp11) and the p30 cleavage product of caspase-11 (Casp11 p30) in WT and Gsdmd−/− mouse RPE cells mock transfected or transfected with Alu RNA. (e) Immunoblots of gasdermin D and the cleavage product of gasdermin D (Gsdmd p30) in mock-transfected and Alu-RNA- transfected human primary RPE cells, WT mouse primary RPE cells, and WT BMDM, as well as in RPE tissue from WT mice subretinally injected with vehicle or Alu RNA. (f) Fundus photographs and immunofluorescence staining of ZO-1 on RPE flat mounts from eyes of Gsdmd−/− mice subjected to reconstitution via in vivo subretinal transfection of empty-vector plasmid (pNull; n = 4 eyes), plasmid expressing WT gasdermin D (pGSDMD-WT; n = 4 eyes) or plasmid expressing mutant gasdermin D incapable of undergoing p30 cleavage (pGSDMD-D276A; n = 5 eyes); the eyes were subretinally injected with Alu RNA. (g) Fundus photographs and immunofluorescence staining of ZO-1 on RPE flat mounts from eyes subretinally injected with Alu RNA from Gsdmd−/− mice subretinally administered vehicle control (vehicle; n = 4 eyes), recombinant mature IL-18 (recIL-18; n = 4 eyes), mature IL-18 expression plasmid (pIL-18ss; n = 5 eyes), or empty-vector control (pNull; n = 4 eyes). (h) GSDMD mRNA abundance, as assessed by RT–qPCR, in RPE tissue of human AMD eyes (n = 7 eyes) and healthy age-matched control eyes (n = 6 eyes). *P = 0.045, two-tailed t test. Data are shown as geometric means ± 95% confidence intervals with individual points plotted. (i) Immunolocalization of gasdermin D in the RPE in human geographic- atrophy eyes and age-matched healthy controls (n = 4 eyes per group). For all immunoblots, cropped gel images of bands of interest of representative immunoblots from three independent experiments and densitometric analysis (mean (s.e.m.)) are shown. In a, f, and g, the degenerated retinal area is outlined by blue arrowheads in the fundus images; loss of regular hexagonal cellular boundaries in ZO-1-stained flat mounts is indicative of degenerated RPE. RPE degeneration was assessed by binary quantification (healthy (%)) and morphometric quantification (PM, polymegethism (mean (s.e.m.))) (Fisher’s exact test for binary; two-tailed t test for morphometry; *P < 0.05; **P < 0.01; ***P < 0.001). For all micrographs, scale bars, 20 µm.

Article Snippet: In vivo transfection of plasmids expressing Alu sequences (pAlu)65,66, emptycontrol vector (pNull), Flag-cGAS (pFlag-cGAS), Flag-GFP, mouse mature IL-18 (pIL-18ss)57,67, WT mouse gasdermin D (pGSDMD-WT), the p30-cleavageincompetent mutant mouse gasdermin D (pGSMDD-D276A)19, IFN-β (Origene, MR226101), or mtDNA (10 ng) was achieved with 10% Neuroporter (Genlantis) as previously described4,5.

Techniques: Injection, Immunofluorescence, Staining, Transfection, Two Tailed Test, Western Blot, In Vivo, Plasmid Preparation, Expressing, Mutagenesis, Control, Recombinant, Quantitative RT-PCR

Journal: Nature medicine

Article Title: cGAS drives noncanonical-inflammasome activation in age-related macular degeneration.

doi: 10.1038/nm.4450

Figure Lengend Snippet: Figure 3 Noncanonical-inflammasome activation and RPE degeneration induced by Alu RNA is mediated by IFN signaling. (a) Top, fundus photographs of eyes of WT (n = 7 eyes) and Ifnar−/− (n = 14 eyes) mice subretinally injected with Alu expression plasmid (pAlu; n = 10 eyes) or empty vector (pNull; n = 11 eyes). Bottom, immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts of the above eyes, showing RPE cell boundaries. (b) Immunoblot of pro-caspase-11 (pro-Casp1) and the p30 cleavage product of caspase-11 (Casp11 p30) in WT and Ifnar−/− mouse RPE cells mock transfected or transfected with Alu RNA. (c) Immunoblot of pro-caspase-4 in IFN-β-treated human RPE cells. (d) IFN-β secretion by human RPE cells transfected with Alu expression plasmid or empty vector. Data presented are mean ± s.d.; n = 3 independent experiments; *P = 0.0012, two-tailed t test. (e) Immunoblot of phosphorylated STAT2 (pSTAT2) and total STAT2 in human RPE cells transfected with Alu expression plasmid or empty vector, or DICER1 or control (ctr) antisense oligonucleotides (AS). (f) Fundus photographs and immunofluorescence staining of ZO-1 on RPE flat mounts from eyes of WT mice coadministerted Alu RNA with IFN-β-neutralizing antibody (n = 6 eyes) or isotype-control IgG (n = 4 eyes). (g) Fundus photographs and immunofluorescence staining of ZO-1 on RPE flat mounts from eyes of WT (n = 6 eyes), Irf3−/− (n = 6 eyes), or Stat2−/− (n = 7 eyes) mice subretinally injected with Alu expression plasmid (n = 9 eyes) or empty vector (n = 10 eyes). (h) Immunolocalization of IFN-β in the RPE in human geographic atrophy eyes and age-matched unaffected controls. Representative images from control and dry AMD eyes are presented; n = 4 eyes per group. (i) Abundance of IFN-β (IFNB1) mRNA in the RPE in human geographic-atrophy eyes compared with age-matched healthy controls; data presented are mean ± s.e.m.; n = 4 eyes; *P = 0.018, two-tailed t test. For all immunoblots, cropped gel images of bands of interest of representative immunoblots of three independent experiments and densitometric analysis (mean (s.e.m.)) are shown. In a, f, and g, the degenerated retinal area is outlined by blue arrowheads in the fundus images; loss of regular hexagonal cellular boundaries in ZO-1-stained flat mounts is indicative of degenerated RPE. RPE degeneration was assessed by binary quantification (healthy (%)) and morphometric quantification (PM, polymegethism (mean (s.e.m.))) (Fisher’s exact test for binary; two-tailed t test for morphometry; *P < 0.05; **P < 0.01; ***P < 0.001). For all micrographs, scale bars, 20 µm.

Article Snippet: In vivo transfection of plasmids expressing Alu sequences (pAlu)65,66, emptycontrol vector (pNull), Flag-cGAS (pFlag-cGAS), Flag-GFP, mouse mature IL-18 (pIL-18ss)57,67, WT mouse gasdermin D (pGSDMD-WT), the p30-cleavageincompetent mutant mouse gasdermin D (pGSMDD-D276A)19, IFN-β (Origene, MR226101), or mtDNA (10 ng) was achieved with 10% Neuroporter (Genlantis) as previously described4,5.

Techniques: Activation Assay, Injection, Expressing, Plasmid Preparation, Immunofluorescence, Staining, Western Blot, Transfection, Two Tailed Test, Control

Journal: Nature medicine

Article Title: cGAS drives noncanonical-inflammasome activation in age-related macular degeneration.

doi: 10.1038/nm.4450

Figure Lengend Snippet: Figure 4 cGAS-driven signaling licenses the noncanonical inflammasome and degeneration of the RPE. (a) Relative abundance of Ifnb1 mRNA in WT and Mb21d1−/− mouse RPE cells mock transfected or transfected with Alu RNA. Data are mean ± s.e.m.; n = 4 cell-culture replicates; *P = 0.0001, two-tailed t test. (b) Immunoblots of pro-caspase-1 (pro-Casp1) and the p20 cleavage product of caspase-1 (Casp1 p20) in WT and Mb21d1−/− mouse RPE cells transfected with Alu expression plasmid (pAlu) or empty-vector control (pNull). (c) Immunoblots of pro-caspase-11 (pro-Casp11) and the p30 cleavage product of caspase-1 (Casp11 p30) in WT and Mb21d1−/− mouse RPE cells transfected with Alu expression plasmid or empty-vector control. (d) IL-18 secretion by WT and Mb21d1−/− mouse RPE cells mock transfected or transfected with Alu RNA. Data are mean ± s.d.; n = 3 independent experiments; *P = 0.032, two-tailed t test. (e) Relative abundance of IFNB1 mRNA in control (shScramble) or cGAS shRNA-knockdown human RPE cells transfected with DICER1 or control (ctr) antisense oligonucleotides (AS). Data are mean ± s.e.m.; n = 3 cell-culture replicates; *P = 0.0002, two-tailed t test. (f) Top, immunoblot of phosphorylated STAT2 (pSTAT2); pro-caspase-4 and caspase-4 p30; pro-caspase-1 and caspase-1 p20 in control (shScramble) or cGAS shRNA-knockdown human RPE cells mock transfected or transfected with Alu RNA. Bottom, immunoblot of cGAS demonstrating knockdown efficiency of cGAS shRNA. (g) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts from eyes of WT (n = 6 eyes) and Mb21d1−/− (n = 8 eyes) mice subretinally injected with vehicle or Alu RNA. (h) Fundus photographs and immunofluorescence staining of ZO-1 on RPE flat mounts from eyes of Mb21d1−/− mice subretinally injected with Alu RNA (n = 7 eyes) and reconstituted with in vivo–transfected cGAS expression plasmid (pFlag-cGAS; n = 4 eyes) or control GFP expression plasmid (pFlag-GFP; n = 3 eyes). (i) Fundus photographs and immunofluorescence staining of ZO-1 on RPE flat mounts from eyes of Mb21d1−/− mice subretinally injected with Alu RNA and with recombinant IFN-β (n = 6 eyes), vehicle control (n = 6 eyes), IFN-β expression plasmid (pIFNB; n = 5 eyes), or empty-vector control (n = 5 eyes). For all immunoblots, cropped gel images of bands of interest of representative immunoblots from three independent experiments and densitometric analysis (mean (s.e.m.)) are shown. In g, h, and i, the degenerated retinal area is outlined by blue arrowheads in the fundus images; loss of regular hexagonal cellular boundaries in ZO-1-stained flat mounts is indicative of degenerated RPE. RPE degeneration was assessed through binary quantification (healthy (%)) and morphometric quantification (PM, polymegethism (mean (s.e.m.))) (Fisher’s exact test for binary; two-tailed t test for morphometry; *P < 0.05; **P < 0.01; ***P < 0.001). For all micrographs, scale bars, 20 µm.

Article Snippet: In vivo transfection of plasmids expressing Alu sequences (pAlu)65,66, emptycontrol vector (pNull), Flag-cGAS (pFlag-cGAS), Flag-GFP, mouse mature IL-18 (pIL-18ss)57,67, WT mouse gasdermin D (pGSDMD-WT), the p30-cleavageincompetent mutant mouse gasdermin D (pGSMDD-D276A)19, IFN-β (Origene, MR226101), or mtDNA (10 ng) was achieved with 10% Neuroporter (Genlantis) as previously described4,5.

Techniques: Transfection, Cell Culture, Two Tailed Test, Western Blot, Expressing, Plasmid Preparation, Control, shRNA, Knockdown, Immunofluorescence, Staining, Injection, In Vivo, Recombinant

Journal: Nature medicine

Article Title: cGAS drives noncanonical-inflammasome activation in age-related macular degeneration.

doi: 10.1038/nm.4450

Figure Lengend Snippet: Figure 5 cGAS in geographic atrophy and RPE degeneration. (a) Immunolocalization of cGAS in the RPE in human geographic-atrophy eyes and age-matched unaffected controls. Representative images from control and dry AMD eyes are presented; n = 4 eyes per group. (b) Immunoblots of pro-caspase-1 and the p20 cleavage product of caspase-1 (Casp1 p20) in WT and Tmem173−/− mouse RPE cells transfected with Alu expression plasmid (pAlu) or empty-vector control plasmid (pNull). (c) Immunoblots of pro-caspase-11 and the p30 cleavage product of caspase-11 (Casp11 p30) in WT and Tmem173−/− mouse RPE cells mock transfected or transfected with Alu RNA. (d) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts from eyes of WT (n = 6 eyes) and Tmem173−/− (n = 10 eyes) mice subretinally injected with vehicle (n = 7 eyes) or Alu RNA (n = 9 eyes). (e) Fundus photographs and immunofluorescence staining of ZO-1 on RPE flat mounts from eyes of Tmem173−/− mice subretinally coadministered Alu RNA with recombinant IFN-β (n = 4 eyes) or vehicle control (n = 4 eyes); or IFN-β expression plasmid (pIFNB; n = 4 eyes) or empty vector control (pNull; n = 4 eyes). For all immunoblots, cropped gel images of bands of interest of representative immunoblots from three independent experiments and densitometric analysis (mean (s.e.m.)) are shown. In d and e, the degenerated retinal area is outlined by blue arrowheads in the fundus images; loss of regular hexagonal cellular boundaries in ZO-1-stained flat mounts is indicative of degenerated RPE. RPE degeneration was assessed by binary quantification (healthy (%)) and morphometric quantification (PM, polymegethism (mean (s.e.m.))) (Fisher’s exact test for binary; two-tailed t test for morphometry; *P < 0.05; **P < 0.01; ***P < 0.001). For all micrographs, scale bars, 20 µm.

Article Snippet: In vivo transfection of plasmids expressing Alu sequences (pAlu)65,66, emptycontrol vector (pNull), Flag-cGAS (pFlag-cGAS), Flag-GFP, mouse mature IL-18 (pIL-18ss)57,67, WT mouse gasdermin D (pGSDMD-WT), the p30-cleavageincompetent mutant mouse gasdermin D (pGSMDD-D276A)19, IFN-β (Origene, MR226101), or mtDNA (10 ng) was achieved with 10% Neuroporter (Genlantis) as previously described4,5.

Techniques: Control, Western Blot, Transfection, Expressing, Plasmid Preparation, Immunofluorescence, Staining, Injection, Recombinant, Two Tailed Test

Journal: Nature medicine

Article Title: cGAS drives noncanonical-inflammasome activation in age-related macular degeneration.

doi: 10.1038/nm.4450

Figure Lengend Snippet: Figure 6 mtDNA in noncanonical-inflammasome activation and RPE degeneration. (a) Relative abundance of cytosolic mtDNA in human RPE cells mock transfected or transfected with Alu RNA. Data are mean ± s.e.m.; n = 3 independent experiments; *P = 0.0018, two-tailed t test. (b) Relative enrichment of mtDNA in cGAS immunoprecipitate in a chromatin-immunoprecipitation-like pulldown assay. Mock- or Alu-RNA-transfected MEFs were analyzed after HA-cGAS immunoprecipitation with anti-HA antibody or isotype-control antibody. Data are mean ± s.e.m.; n = 3; *P = 0.008, two-tailed t test. (c) Relative abundance of cytosolic mtDNA in WT and Ppif−/− mouse RPE cells mock transfected (n = 4 cell-culture replicates) or transfected with Alu RNA. Data are mean ± s.e.m.; n = 6 cell-culture replicates; *P = 0.004, two-tailed t test. (d) Fundus photographs and immunofluorescence staining of zonula occludens-1 (ZO-1) on RPE flat mounts from eyes of WT (n = 6 eyes) and Ppif−/− (n = 12 eyes) mice subretinally injected with vehicle (n = 9 eyes) or Alu RNA (n = 9 eyes). (e) Immunoblot for procaspase-1 (pro-Casp1) and the p20 cleavage product of caspase-1 in WT and Ppif−/− mouse RPE cells mock transfected or transfected with Alu RNA. (f) Immunoblot for procaspase-11 (pro-Casp11) and the p30 cleavage product of caspase-11 (Casp11 p30) in WT and Ppif−/− mouse RPE cells mock transfected or transfected with Alu RNA. (g) Immunoblot for procaspase-4 (pro-Casp4) and the p30 cleavage product of caspase-4 (Casp4 p30) in WT and mtDNA-deficient Rho0 ARPE19 human RPE cells mock transfected or transfected with Alu RNA. (h,i) IL-18 secretion (data are mean ± s.d.; n = 4 independent experiments; *P = 0.0001, two-tailed t test) (h) and IFN-β secretion (data are mean ± s.d.; n = 4 independent experiments; *P = 0.004, two-tailed t test) (i) from WT and mtDNA-deficient Rho0 ARPE19 human RPE cells mock transfected or transfected with Alu RNA. (j) Fundus photographs and immunofluorescence staining of ZO-1 on RPE flat mounts from eyes of Ppif−/− mice subretinally coadministered Alu RNA with recombinant IFN-β (n = 5 eyes) or vehicle control (n = 5 eyes); or IFN-β expression plasmid (pIFNB; n = 6 eyes) or empty-vector control (pNull; n = 5 eyes). For all immunoblots, cropped gel images of bands of interest of representative immunoblots from three independent experiments and densitometric analysis (mean (s.e.m.)) are shown. In d and j, the degenerated retinal area is outlined by blue arrowheads in the fundus images; loss of regular hexagonal cellular boundaries in ZO-1-stained flat mounts is indicative of degenerated RPE. RPE degeneration was assessed by binary quantification (healthy (%)) and morphometric quantification (PM, polymegethism (mean (s.e.m.))) (Fisher’s exact test for binary; two-tailed t test for morphometry; *P < 0.05; **P < 0.01; ***P < 0.001). For all micrographs, scale bars, 20 µm.

Article Snippet: In vivo transfection of plasmids expressing Alu sequences (pAlu)65,66, emptycontrol vector (pNull), Flag-cGAS (pFlag-cGAS), Flag-GFP, mouse mature IL-18 (pIL-18ss)57,67, WT mouse gasdermin D (pGSDMD-WT), the p30-cleavageincompetent mutant mouse gasdermin D (pGSMDD-D276A)19, IFN-β (Origene, MR226101), or mtDNA (10 ng) was achieved with 10% Neuroporter (Genlantis) as previously described4,5.

Techniques: Activation Assay, Transfection, Two Tailed Test, Chromatin Immunoprecipitation, Immunoprecipitation, Control, Cell Culture, Immunofluorescence, Staining, Injection, Western Blot, Recombinant, Expressing, Plasmid Preparation