Journal: Immunity, Inflammation and Disease

Article Title: Upregulation of IFNE in cervical biopsies of patients with high‐risk human papillomavirus infections

doi: 10.1002/iid3.1111

Figure Lengend Snippet: IFN‐ε is expressed at high levels in cervical tissue regardless of hormonal status. (Α) Cervical biopsy samples ( n = 32) were examined for mRNA levels of IFN‐α2, IFN‐β, IFN‐ε, IFN‐κ, IFN‐ω, IFN‐γ, and IFN‐λ1. (B) IFN expression levels in cervical samples from patients in follicular ( n = 12) and luteal ( n = 13) stage and postmenopausal patients ( n = 3). IFN expression of postpartum patients and patients undergoing GnRH‐α therapy is not shown.

Article Snippet: For immunofluorescence, sections were blocked with 10% goat serum and incubated with IFN‐ε antibody (1 μg/mL, Novus biologicals) or mouse IgG2b (1 μg/mL, BioLengends) for 24 h at 4°C.

Techniques: Expressing

Journal: Immunity, Inflammation and Disease

Article Title: Upregulation of IFNE in cervical biopsies of patients with high‐risk human papillomavirus infections

doi: 10.1002/iid3.1111

Figure Lengend Snippet: IFN‐ε expression is upregulated in HR‐HPV positive patients at the mRNA but not protein level. (A) IFN expression levels in cervical samples from HR‐HPV negative ( n = 10) and HR‐HPV positive ( n = 22) patients (* p < .05). (B) IFN‐ε expression determined in representative samples of HR‐HPV negative ( n = 10) and HR‐HPV positive ( n = 22) patients by immunoblotting. (C) Immunofluorescence staining for IFN‐ε (red) and DAPI (blue) in cervical tissues of HR‐HPV negative and HR‐HPV positive patients. (D) Quantitative analysis of immunofluorescence staining for IFN‐ε in cervical samples of HR‐HPV negative ( n = 4) and HR‐HPV positive ( n = 6) patients. (E) Immunohistochemical analysis of IFN‐ε protein levels in cervical biopsies of HR‐HPV positive and HR‐HPV negative patients.

Article Snippet: For immunofluorescence, sections were blocked with 10% goat serum and incubated with IFN‐ε antibody (1 μg/mL, Novus biologicals) or mouse IgG2b (1 μg/mL, BioLengends) for 24 h at 4°C.

Techniques: Expressing, Western Blot, Immunofluorescence, Staining, Immunohistochemical staining

Journal: PLOS Pathogens

Article Title: TRIM29 knockout pigs exhibit enhanced broad-spectrum disease resilience by amplifying type I interferon antiviral defenses

doi: 10.1371/journal.ppat.1014023

Figure Lengend Snippet: A. Total RNA was isolated from PK15 cells infected with PRV at MOI = 0.1 for 24 h and the relative mRNA expression of TRIM29 was detected by qPCR with GAPDH as the internal gene control. B. Representative immunoblot analysis of TRIM29 and β-actin in PK15 cells infected with PRV at MOI = 0.1 for 24 h. C. Quantification of protein levels by analyzing band density shown in B using Image J software. D. The relative mRNA expression of IFNβ was detected by qPCR with GAPDH as the internal gene control in PK15 cells infected with PRV at MOI = 0.1 for 24 h. E. Three pig TRIM29-specific siRNA were design for gene silencing in PK15 cells. The TRIM29 mRNA levels were analyzed by qPCR in PK15 cells transfected with the indicated siRNA for 36 h. F. Representative immunoblot analysis of TRIM29 protein in siRNA-transfected PK15 cells. G. Quantification of TRIM29 protein levels shown in F by analyzing band density. H. qPCR of IFNβ mRNA in PK15 cells transfected with si882 and control siNC for 24 h and then infected with PRV at MOI = 0.1 or treated with cGAMP for 24 h. I. PRV DNA genome copies were determined in culture supernatant of PK15 cells transfected with si882 and NC siRNA for 24 h and then infected with PRV at MOI = 0.1 for 24 h. J. PRV viral titers determined by TCID 50 in the TRIM29-knockdown PK15 with PRV infection. K. Representative immunoblot of TRIM29 protein in PK15 cells transfected with TRIM29-overexpressing plasmid and blank vector as the control. L. Quantification of overexpressing TRIM29 by measuring band density. M. The TRIM29-overexpressing PK15 were infected with PRV at MOI = 0.1 or treated with cGAMP for 24 h and the relative mRNA expression of IFNβ was detected by qPCR. N. PRV DNA copies were determined in the TRIM29-overexpressing PK15 with PRV infection. O. PRV viral titers determined by TCID 50 in the TRIM29-overexpressing PK15 with PRV infection. P. PRV growth curve in TRIM29-knockdown and control PK15 cells inoculated at MOI = 0.1. Q. PRV growth curve in TRIM29-knockdown and control PK15 cells inoculated at MOI = 1. R. PRV growth curve in TRIM29-overexpressing and control PK15 cells inoculated at MOI = 0.1. S. PRV growth curve in TRIM29-overexpressing and control PK15 cells inoculated at MOI = 1. T. Immunoblot analysis of TRIM29, cGAS, STING, TBK1, phosphorylated TBK1 (pTBK1), IRF3, phosphorylated IRF3 (pIRF3), and GAPDH in PK15 cells transfected with TRIM29 siRNA (si882, left panel) and overexpressing vector (right panel) with mock and PRV infection. Data are expressed as mean ± SD. The p-values between the indicated groups were calculated using a two-tailed unpaired t-test (A, C, D, H-J, L and N-S) or one-way ANOVA with Sidak’s multiple comparison test (E and G) to determine statistical significance.

Article Snippet: The following primary antibodies used in this study, anti-TRIM29 (1:1000; 17542–1-AP; Proteintech), anti-cGAS (1:2000; 26416–1-AP; Proteintech), anti-MAVS (1:2000; 14341–1-AP; Proteintech), anti-STING (1:1000; 19851–1-AP; Proteintech), anti-TBK1 (1:1000; #3504; Cell Signaling Technology), anti-phospho TBK1 (1:1000; #5483; Cell Signaling Technology), anti-IRF3 (1:5000; 11312–1-AP; Proteintech), anti-phospho IRF3 (1:1000; 29528–1-AP; Proteintech), anti-IL-1β (1:500; P420B; Thermo Fisher Scientific), anti-COX2 (1:1000; EPR12012 ; Abcam), anti-TNFα (1:500; GTX110520; GeneTex), anti-β-actin (1:5000; 4970; Cell Signaling Technology) and anti-GAPDH (1:5000; 2118; Cell Signaling Technology).

Techniques: Isolation, Infection, Expressing, Control, Western Blot, Software, Transfection, Knockdown, Plasmid Preparation, Two Tailed Test, Comparison

Journal: PLOS Pathogens

Article Title: TRIM29 knockout pigs exhibit enhanced broad-spectrum disease resilience by amplifying type I interferon antiviral defenses

doi: 10.1371/journal.ppat.1014023

Figure Lengend Snippet: A. The relative mRNA expression of TRIM29 was determined by qPCR in PK15 cells with mock and VSV infection at MOI = 0.1 for 24 h. B. The relative mRNA expression of IFNβ was determined by qPCR in PK15 cells with mock and VSV infection at MOI = 0.1 for 24 h. C. Determination of IFNβ level in TRIM29-knockdown PK15 cells infected with VSV at MOI = 0.1 or treated with poly (I:C) for 24 h. D. Determination of VSV RNA copies in the supernatant of TRIM29-knockdown cells infected with VSV at MOI = 0.1 for 24 h. E. Determination of VSV titers in TRIM29-knockdown cells infected with VSV at MOI = 0.1 for 24 h. F. The relative mRNA expression of IFNβ was determined by qPCR in TRIM29-overexpressing PK15 cells with mock and VSV infection at MOI = 0.1 for 24 h or treated with poly (I:C) for 24 h. G. Determination of VSV RNA copies in the supernatant of TRIM29-overexpressing cells. H. Determination of VSV titers in the supernatant of TRIM29-overexpressing cells. I. EGFP fluorescence showing VSV infection level in TRIM29-knockdown PK15 cells infected with VSV at MOI = 0.1 for 24 h. J. EGFP fluorescence in TRIM29-overexpressing PK15 cells infected with VSV at MOI = 0.1 for 24 h. Mock represents uninfected groups. Scale bars, 100 μm. K. VSV growth curve in TRIM29-knockdown and control PK15 cells inoculated at MOI = 0.1. L. VSV growth curve in TRIM29-knockdown and control PK15 cells inoculated at MOI = 1. M. VSV growth curve in TRIM29-overexpressing and control PK15 cells inoculated at MOI = 0.1. N. VSV growth curve in TRIM29-overexpressing and control PK15 cells inoculated at MOI = 1. O. Immunoblot analysis of TRIM29, MAVS, STING, TBK1, pTBK1, IRF3, pIRF3, and GAPDH in PK15 cells transfected with si882 (left panel) and TRIM29-overexpressing vector (right panel) with mock and VSV infection. Data are expressed as mean ± SD. The p-values between the indicated groups were calculated using a two-tailed unpaired t-test (A-H and K-N).

Article Snippet: The following primary antibodies used in this study, anti-TRIM29 (1:1000; 17542–1-AP; Proteintech), anti-cGAS (1:2000; 26416–1-AP; Proteintech), anti-MAVS (1:2000; 14341–1-AP; Proteintech), anti-STING (1:1000; 19851–1-AP; Proteintech), anti-TBK1 (1:1000; #3504; Cell Signaling Technology), anti-phospho TBK1 (1:1000; #5483; Cell Signaling Technology), anti-IRF3 (1:5000; 11312–1-AP; Proteintech), anti-phospho IRF3 (1:1000; 29528–1-AP; Proteintech), anti-IL-1β (1:500; P420B; Thermo Fisher Scientific), anti-COX2 (1:1000; EPR12012 ; Abcam), anti-TNFα (1:500; GTX110520; GeneTex), anti-β-actin (1:5000; 4970; Cell Signaling Technology) and anti-GAPDH (1:5000; 2118; Cell Signaling Technology).

Techniques: Expressing, Infection, Knockdown, Fluorescence, Control, Western Blot, Transfection, Plasmid Preparation, Two Tailed Test