Journal: Investigative Ophthalmology & Visual Science

Article Title: PIM1 Inhibition Alleviates Aspergillus fumigatus Keratitis by Regulating DDX41-Mediated STING Signaling Pathway

doi: 10.1167/iovs.67.4.2

Figure Lengend Snippet: PIM1 directly interacts with DDX41 and regulates its phosphorylation. ( A ) HCECs transfected with either pcDNA3.1 (Flag) or pcDNA3.1 PIM1 (Flag-PIM1) were stimulated with A. fumigatus hyphae (1 × 10 6 hyphal fragments/mL) for 12 hours. Cell lysates were immunoprecipitated using anti-Flag beads, and bound proteins were separated by SDS-PAGE and visualized by silver staining. ( B ) A GST pull-down assay was performed to examine direct binding between PIM1 and DDX41. ( C ) Endogenous interaction between PIM1 and DDX41 in HCECs was assessed by co-IP. HEK293T cells were cotransfected with Flag-PIM1 and Myc-DDX41 for 48 hours. (D) Co-IP was carried out with anti-Flag beads, and Myc-DDX41 was detected with anti-Myc antibody. ( E ) Reciprocal co-IP was performed using anti-Myc beads, and Flag-PIM1 was probed with anti-Flag antibody. ( F ) HCECs were transfected with NC siRNA (siNC) or PIM1 siRNAs (siPIM1-1 and siPIM1-2) for 24 hours, then exposed to A. fumigatus hyphae for 12 hours. DDX41 was immunoprecipitated and immunoblotted with anti–p-Ser and DDX41 antibodies. ( G ) HCECs were transfected with pcDNA3.1 (Vector) or pcDNA3.1-PIM1 (PIM1) for 24 hours, then exposed to A. fumigatus hyphae for 12 hours, followed by DDX41 immunoprecipitation and immunoblotting with indicated antibodies. ( H ) HCECs were transfected with NC siRNA (siNC), PIM1 siRNA (siPIM1-2), and/or pcDNA3.1-PIM1 (PIM1) for 24 hours, then treated with A. fumigatus hyphae for 12 hours. Co-IP of DDX41 was performed, and associated proteins were analyzed by Western blot. ( I ) Quantification of protein levels in ( H ). ( J ) HCECs were pretreated with SGI-1776 (0 and 4 µM) for 12 hours prior to 12-hour stimulation with A. fumigatus hyphae. Co-IP assay was performed using anti-DDX41 antibody and immunoblotted with the antibodies indicated. ( K ) Quantification of protein levels in ( J ). ( L ) Recombinant His-PIM1 and GST-DDX41 were incubated in reaction buffer with or without phosphatase for 30 minutes. The protein levels of p-Ser, His-PIM1, and GST-DDX41 were detected by Western blot. Quantification of protein levels in F, G, and L is shown in A–C. Data are presented as the mean ± SD; ** P < 0.01; n = 3.

Article Snippet: Antibodies recognizing phosphorylated STING (Ser366, 19781; Ser365, 72971; 1:1000), phosphorylated TBK1 (5483, 1:1000), phosphorylated IRF3 (4947, 1:1000), and DDX41 (15076, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Transfection, Immunoprecipitation, SDS Page, Silver Staining, Pull Down Assay, Binding Assay, Co-Immunoprecipitation Assay, Plasmid Preparation, Western Blot, Recombinant, Incubation

Journal: Investigative Ophthalmology & Visual Science

Article Title: PIM1 Inhibition Alleviates Aspergillus fumigatus Keratitis by Regulating DDX41-Mediated STING Signaling Pathway

doi: 10.1167/iovs.67.4.2

Figure Lengend Snippet: PIM1 regulates STING signaling through DDX41. ( A ) HCECs were transfected with pcDNA3.1 (Vector) or pcDNA3.1-PIM1 (PIM1) for 24 hours, followed by stimulation with A. fumigatus hyphae (1 × 10 6 hyphal fragments/mL) for 12 hours. Protein levels of p-STING, STING, p-TBK1, TBK1, p-IRF3, IRF3, PIM1, and β-actin were assessed by Western blot. ( B ) HCECs were transfected with NC siRNA (siNC) or PIM1 siRNAs (siPIM1-1 and siPIM1-2) for 24 hours, then treated with A. fumigatus hyphae for 12 hours. Protein expression was analyzed via Western blot. ( C ) Quantification of protein levels in ( A ). ( D ) Quantification of protein levels in ( B ). ( E ) HCECs were transfected with NC siRNA (siNC), PIM1 siRNA-2 (siPIM1-2), and/or pcDNA3.1-PIM1 (PIM1) for 24 hours, followed by A. fumigatus hyphae treatment for 12 hours. Protein levels were detected by Western blot. ( F ) HCECs were pretreated with SGI-1776 (0, 2, 4 µM) for 12 hours, then exposed to A. fumigatus hyphae for 12 hours. Protein levels were detected by Western blot. ( G ) HCECs were transfected with NC siRNA (siNC), PIM1 siRNA-2 (siPIM1-2), DDX41 siRNA (siDDX41), or pcDNA3.1-DDX41 (DDX41) for 24 hours, followed by A. fumigatus hyphae treatment for 12 hours. Protein levels were detected by Western blot. ( H ) Quantification of protein levels in ( E ). ( I ) Quantification of protein levels in ( F ). Quantification of protein levels in G is provided in D. Data are presented as the mean ± SD; ** P < 0.01; n = 3.

Article Snippet: Antibodies recognizing phosphorylated STING (Ser366, 19781; Ser365, 72971; 1:1000), phosphorylated TBK1 (5483, 1:1000), phosphorylated IRF3 (4947, 1:1000), and DDX41 (15076, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: PIM1 Inhibition Alleviates Aspergillus fumigatus Keratitis by Regulating DDX41-Mediated STING Signaling Pathway

doi: 10.1167/iovs.67.4.2

Figure Lengend Snippet: PIM1 regulates A. fumigatus –induced inflammatory response by interacting with DDX41. HCECs were transfected with NC siRNA (siNC), PIM1 siRNA-2 (siPIM1-2), DDX41 siRNA (siDDX41), and/or pcDNA3.1-DDX41 (DDX41) for 24 hours, followed by stimulation with A. fumigatus hyphae (1 × 10 6 hyphal fragments/mL) for 12 hours. ( A ) Protein levels of DDX41, PIM1, and β-actin were analyzed by Western blot. Quantitative data are shown in A. ( B ) mRNA expression levels of TNF-α , IL-6 , IL-1β , and IFN-β were evaluated by qRT-PCR. ( C ) Secreted levels of TNF-α, IL-6, IL-1β, and IFN-β in culture supernatants were measured using ELISA. HCECs were transfected with pcDNA3.1 (Vector) or pcDNA3.1-PIM1 (PIM1) for 24 hours and then treated with A. fumigatus hyphae for 12 hours. C176 (1 µM) was added to the medium for 24 hours before harvest. ( D ) Protein levels of PIM1 and β-actin were analyzed by Western blot. Quantitative analysis is shown in B. ( E ) mRNA expression levels of TNF-α , IL-6 , IL-1β , and IFN-β were evaluated by qRT-PCR. ( F ) Secreted levels of TNF-α, IL-6, IL-1β, and IFN-β in culture supernatants were measured using ELISA. Data are presented as the mean ± SD; * P < 0.05, ** P < 0.01; n = 3.

Article Snippet: Antibodies recognizing phosphorylated STING (Ser366, 19781; Ser365, 72971; 1:1000), phosphorylated TBK1 (5483, 1:1000), phosphorylated IRF3 (4947, 1:1000), and DDX41 (15076, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: Investigative Ophthalmology & Visual Science

Article Title: PIM1 Inhibition Alleviates Aspergillus fumigatus Keratitis by Regulating DDX41-Mediated STING Signaling Pathway

doi: 10.1167/iovs.67.4.2

Figure Lengend Snippet: Knockdown of PIM1 ameliorates A. fumigatus keratitis in mice. ( A ) Mouse corneas were infected with 5 µL of live A. fumigatus hyphae (1 × 10 8 hyphal fragments/mL) for 0.5, 1, and 3 days, then excised for analysis. Protein levels of p-STING, STING, PIM1, and β-actin were assessed by Western blot. Quantitative data are provided in A. ( B ) DDX41 was immunoprecipitated from corneal lysates and immunoblotted with anti–p-Ser and anti-DDX41 antibodies. Quantification is shown in B. Mice received subconjunctival injection of 5 µL control siRNA (siNC, 10 µM) or PIM1 siRNAs (siPIM1-1/siPIM1-2, 10 µM). After 24 hours, corneas were infected with 5 µL of live hyphae for an additional 24 hours before harvesting. ( C ) Keratitis severity was evaluated by slit-lamp examination. ( D ) Clinical scores were calculated based on slit-lamp observations. ( E ) Fungal burden was quantified by colony-forming unit (CFU) assays. ( F ) Protein expression of p-STING, STING, PIM1, and β-actin in mouse corneas was detected by Western blot. Quantitative results are shown in C. ( G ) DDX41 was immunoprecipitated and immunoblotted with anti–p-Ser and anti-DDX41 antibodies. Quantification is presented in D. ( H ) mRNA expression of TNF-α , IL-6 , IL-1β , and IFN-β was measured by qRT-PCR. ( I ) Secreted levels of TNF-α, IL-6, IL-1β, and IFN-β in corneal homogenates were detected using ELISA. Data are presented as the mean ± SD; ** P < 0.01; n = 6.

Article Snippet: Antibodies recognizing phosphorylated STING (Ser366, 19781; Ser365, 72971; 1:1000), phosphorylated TBK1 (5483, 1:1000), phosphorylated IRF3 (4947, 1:1000), and DDX41 (15076, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Knockdown, Infection, Western Blot, Immunoprecipitation, Injection, Control, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: PIM1 Inhibition Alleviates Aspergillus fumigatus Keratitis by Regulating DDX41-Mediated STING Signaling Pathway

doi: 10.1167/iovs.67.4.2

Figure Lengend Snippet: PIM1 inhibitor inhibits A. fumigatus keratitis in vivo. Mouse corneas were infected with 5 µL of live A. fumigatus hyphae (1 × 10 8 hyphal fragments/mL) for 24 hours. Two hours prior to infection, mice received a subconjunctival injection of 5 µL SGI-1776 (1 µg/µL or 2 µg/µL). Corneas were harvested 24 hours postinfection. ( A ) Keratitis severity was evaluated by slit-lamp examination. ( B ) Clinical scores were calculated based on slit-lamp observations. ( C ) Fungal burden was quantified by CFU assays. (D) Protein expression of p-STING, STING, PIM1, and β-actin in mouse corneas was analyzed by Western blot. Quantification is presented in A. ( E ) DDX41 was immunoprecipitated from corneal lysates and immunoblotted with anti–p-Ser and anti-DDX41 antibodies. Quantification is provided in B. ( F ) mRNA expression levels of TNF-α , IL-6 , IL-1β , and IFN-β were measured by qRT-PCR. ( G ) Protein levels of TNF-α, IL-6, IL-1β, and IFN-β in corneal homogenates were determined using ELISA. Data are presented as the mean ± SD; * P < 0.05, ** P < 0.01; n = 6.

Article Snippet: Antibodies recognizing phosphorylated STING (Ser366, 19781; Ser365, 72971; 1:1000), phosphorylated TBK1 (5483, 1:1000), phosphorylated IRF3 (4947, 1:1000), and DDX41 (15076, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: In Vivo, Infection, Injection, Expressing, Western Blot, Immunoprecipitation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: PIM1 Inhibition Alleviates Aspergillus fumigatus Keratitis by Regulating DDX41-Mediated STING Signaling Pathway

doi: 10.1167/iovs.67.4.2

Figure Lengend Snippet: Working model of the PIM1-DDX41-STING axis in A. fumigatus keratitis. A. fumigatus infection upregulates PIM1 expression. PIM1 then directly binds to and phosphorylates the innate immune sensor DDX41. Phosphorylated DDX41 activates the STING signaling pathway, leading to the phosphorylation of TBK1 and IRF3. This cascade ultimately promotes the transcription and secretion of proinflammatory cytokines (TNF-α, IL-6, IL-1β, IFN-β), contributing to the immunopathology of fungal keratitis. Pharmacologic inhibition of PIM1 by SGI-1776 disrupts this axis, attenuating DDX41/STING activation and the subsequent inflammatory response.

Article Snippet: Antibodies recognizing phosphorylated STING (Ser366, 19781; Ser365, 72971; 1:1000), phosphorylated TBK1 (5483, 1:1000), phosphorylated IRF3 (4947, 1:1000), and DDX41 (15076, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Infection, Expressing, Phospho-proteomics, Inhibition, Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: IFN-β expression is directly activated in human neutrophils transfected with plasmid DNA and is further increased via TLR-4-mediated signaling.

doi: 10.4049/jimmunol.1102985

Figure Lengend Snippet: FIGURE 6. Neutrophil recognition of intracellular plasmid DNA is dependent on IFI16. (A) Whole neutrophil and monocyte extracts (60 mg) were electrophoresed and immunoblotted using Abs specific for IFI16, LRRFIP1, DDX41 RIG-I, STING, and tubulin. (B) Whole neutrophil lysates (500 mg), prepared using the ChIP assay buffer, were subjected to immunoprecipitation with IFI16, LRRFIP1, DDX41, or anti-mouse IgG1 (control) Abs. Representative immunoblot experiments are shown (n = 3) for (A) and (B). (C) Neutrophils were transfected with pEGFP, cultured for 3 and 5 h, and then disrupted by ChIP assay buffer. Whole-cell lysates were then subjected to immunoprecipitation with IFI16, LRRFIP1, DDX41, or anti-mouse IgG1 control Abs. Finally, coimmunoprecipitated DNA was purified and analyzed by qPCR using two sets of primers specific for pEGFP (pEGFP-backbone and pEGFP-GFP). Data from qPCR are expressed as percentages over input DNA. Results are from one experi- ment representative of four.

Article Snippet: PKCε (sc-214), p-PKCε (Ser729; sc-12355), PKCbII (sc-210), IRF3 (sc9082), NF-kB p65 (sc-372), NF-kB p50 (sc-7178), IkB-a (sc-371), IFNinducible protein 16 (IFI16; sc-8023), DDX41 (sc-166225), and leucinerich repeat (in Flightless I) interacting protein-1 (LRRFIP1; sc-135917) Abs were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Plasmid Preparation, Immunoprecipitation, Control, Western Blot, Transfection, Cell Culture

Journal: Cells

Article Title: Cytosolic Immunostimulatory DNA Ligands and DNA Damage Activate the Integrated Stress Response, Stress Granule Formation, and Cytokine Production

doi: 10.3390/cells15020139

Figure Lengend Snippet: The cGAS-STING signaling pathway is required for SG induction by DNA ligands. ( A ) HT1080 cells were treated with siRNA for 36 h to knockdown DDX41, IFI16, or cGAS, followed by transfection with pdA:dT (5 μg/mL) or HT-DNA (4 μg/mL) for 6 h, and cell lysates were analyzed for phosphorylation of STING and TBK1 compared to total STING and total TBK1 normalized to β-actin levels. The ratios of p-STING/total STING and p-TBK1/total TBK1 were quantified by Image J. The efficiency of knockdown by siRNA was compared to control cells and determined by immunoblot analysis. ( B ) WT, cells expressing HA-STING, or STING KO HT1080 cells. ( C ) WT, RIG-I KO, and STING KO HT1080 cells were transfected with the indicated STING ligands for 8 h or indicated times and cell lysates were probed with indicated antibodies on immunoblots; two exposures of STING (L, low and H, high) of the same blot are shown. The ratios of the indicated phospho-proteins/total proteins for the antibodies probed were quantified by Image J. ( D ) WT and STING KO HT1080 cells transfected with pdA:dT (5 μg/mL) or HT-DNA (4 μg/mL) and SG were immunostained with anti-G3BP1, and SG positive cells were quantitated. Percentage of cells showing G3BP1-positive SG puncta was determined from at least 100 cells from three replicates. Results are mean ± SD. ( E ) WT and G3BP1KO HT1080 cells were transfected with pdA:dT (5 μg/mL) or mock-transfected and after 8 h were immunostained for SG markers G3BP1 (red) and TIA1 (green) and nuclei (DAPI, blue), and SG positive cells were analyzed by confocal microscopy. The magnified images correspond to boxed regions. Expression of STING or G3BP1 in KO cells was compared to WT cells by immunoblot analysis. Scale bars, 20 µm. The results presented here are representative of at least three biological repeats. *** p < 0.001.

Article Snippet: G3BP1 (sc-81940) and TIA1 (sc-166247) were from Santa Cruz Biotechnology (Dallas, TX, USA). cGAS (26416-1-AP) and BiP (66574-1-Ig) were from Proteintech (Rosemont, IL, USA). siRNA towards DDX41 (sc-91765), IFI16 (sc-35633), and cGAS (sc-95512) were from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Knockdown, Transfection, Phospho-proteomics, Control, Western Blot, Expressing, Confocal Microscopy