Journal: PLOS Pathogens

Article Title: Microtubule disruption synergizes with STING signaling to show potent and broad-spectrum antiviral activity

doi: 10.1371/journal.ppat.1012048

Figure Lengend Snippet: (A-L) THP1-Lucia ISG (WT and STING KO) cells were treated with cGAMP with or without indicated doses of MMAE for 24 h (A-F) or 6 h (J-L), and the fold change of luminescence was normalized to DMSO-treated cells. The activation of STING pathway was analyzed by immunoblotting (G-L). (M) THP1-Lucia ISG cells were pretreated for 12 h with or without anti-IFNAR2 antibody (20 μg/ml), and then stimulated with cGAMP or IFNβ (200 pg/ml) for 24 h in the absence or presence of MMAE (1 μM). Fold change of luminescence was normalized to DMSO-treated cells. (N and O) THP1-Lucia ISG (WT and IRF3 KO) cells were treated with cGAMP and/or indicated doses of MMAE for 12 h (N) or 6 h (O). IFNβ production was measured by ELISA analysis (N). Expression of IRF3 and the activation of STING pathway was analyzed by immunoblotting (N and O).

Article Snippet: Rabbit antibody against IRF3 (11312-1-AP) was purchased from proteintech.

Techniques: Activation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing

Journal: PLOS Pathogens

Article Title: Microtubule disruption synergizes with STING signaling to show potent and broad-spectrum antiviral activity

doi: 10.1371/journal.ppat.1012048

Figure Lengend Snippet: MMAE changed cGAMP-mediated STING trafficking routes from ER to Golgi apparatus by disrupting the microtubule network, and delayed the trafficking-mediated STING degradation. MMAE dispersed the cGAMP-mediated STING perinuclear puncta into large number of tiny vesicles throughout the cytoplasm. The accumulated STING vesicles further amplified the cGAMP-mediated TBK1-STING-IRF3 signaling cascade, and promoted the production of IFNs and ISGs expression. MMAE alone restricted viral replication and infection by destroying microtubule networks, while MMAE combined with cGAMP exerted potent and broad-spectrum antiviral activity in vitro and in vivo in a STING-dependent manner.

Article Snippet: Rabbit antibody against IRF3 (11312-1-AP) was purchased from proteintech.

Techniques: Amplification, Expressing, Infection, Activity Assay, In Vitro, In Vivo

Journal: Nature Communications

Article Title: Regulation of TRIF-mediated innate immune response by K27-linked polyubiquitination and deubiquitination

doi: 10.1038/s41467-019-12145-1

Figure Lengend Snippet: USP19 negatively regulates TLR3/4-mediated signaling in primary cells. a Effects of USP19-deficiency on poly(I:C)- and LPS-induced transcription of downstream genes. Usp19 +/+ and Usp19 −/− BMDMs were stimulated with poly(I:C) (50 μg/ml), LPS (50 ng/ml) for the indicated times before qPCR with the indicated gene primers was performed. Graphs show mean ± SD; n = 3 independent samples. * P < 0.05, ** P < 0.01, *** P < 0.001 (unpaired t- test). b Effects of USP19-deficiency on poly(I:C)- and LPS-induced phosphorylation of TBK1, p65, IRF3, and ΙκΒα. Usp19 +/+ and Usp19 −/− BMDMs were stimulated with poly(I:C) (100 μg/ml) or LPS (100 ng/ml) for the indicated times before immunoblots were performed with the indicated antibodies. Data are representative of three experiments with similar results. Source data are provided as a Source Data file. Error bars represent standard deviation of the mean

Article Snippet: Mouse monoclonal antibodies against Flag (Origene, 1:2000, F3165), HA (Origene, 1:2000, H6908), β-actin (Sigma, 1:10,000, A2228), Myc (CST, 1:1000, 5605), p-IκBα (CST, 1:1000, 9246 L); rabbit antibodies against p-IRF3 (CST, 1:500, 37829), USP19 (Abcam, 1:1000, ab93159), TRIF (Abcam, 1:1000, ab180689), p65 (Santa Cruz Biotechnology, 1:1000, 71675), p-p65(S536) (CST, 1:1000, 3033),TBK1 (Abcam, 1:1000, ab40676) and p-TBK1 (Abcam, 1:1000, ab109272), ubiquitin (Abcam, 1:500, ab134953), K27-linkage specific polyubiquitin (Abcam, 1:1000, 181537), TLR3 (CST, 1:500, 6961), TLR4 (R&D, 1:500, AF1478), TRAM (Abcam, 1:500, ab96106), KCTD10 (Proteintech, 1:1000, 27279–1-AP); poly(I:C) (Invivogen), LPS (Sigma), R848 (Invivogen), PGN (Invivogen), human IFN-γ (Peprotech), murine M-CSF (Peprotech), Trizol (Takara Bio), SYBR Green (BIO-RAD), dual-specific luciferase assay kit (Promega, E1980), polybrene (Millipore,TR-1003-G), type II collagenase (Worthington), DNase I (Sigma-Aldrich), and d -galactosamine hydrochloride (D-Gal) (Sigma); and ELISA kits for TNF (Biolegend), IL-6 (Biolegend), CXCL10 (Boster) and IFN-β (PBL) were purchased from the indicated companies.

Techniques: Phospho-proteomics, Western Blot, Standard Deviation

Journal: Microbiology Spectrum

Article Title: TRIM56-mediated production of type I interferon inhibits intracellular replication of Rickettsia rickettsii

doi: 10.1128/spectrum.03695-23

Figure Lengend Snippet: TRIM56 enhances the R. rickettsii -induced cGAS-STING signaling through ubiquitination of STING. ( A–B ) PMA-differentiated WT and trim56 −/− THP-1 cells were infected by R. rickettsii, and then, the infected cells were collected at 0, 0.5, 2, 4, 6, and 12 hours post-infection for Western blotting analysis, followed by probing with anti-p-IRF3, anti-IRF3, and anti-GAPDH, respectively. ( B ) Densitometry of ( A ) was presented as a fold change between the ratios of p-IRF3/IRF3 ( n = 3, mean ± SD). (C–D) PMA-differentiated WT and trim56 –/− THP-1 cells were infected by R. rickettsii, and then, the infected cells were collected for Western blotting analysis, followed by probing with anti-p-STING, anti-STING, and GAPDH, respectively ( C ). Densitometry of ( C ) was presented as a fold change between the ratios of p-STING/STING ( n = 3, mean ± SD) ( D ). (E–F ) WT and trim56 –/– HeLa cells transfected with STING-FLAG and HA-Ub plasmids were infected with R. rickettsii at an MOI of 1 for 2 days, and then, the protein complexes were extracted from the cell lysates with anti-FLAG M2 beads. The immunoprecipitation with FLAG lysates ( E ) and whole-cell lysates ( F ) was probed with FLAG and HA antibodies. Data presented were representative of at least three independent experiments. ns P ≥ 0.05; ** P < 0.01; **** P < 0.0001.

Article Snippet: The primary antibodies used are as follows: rabbit polyclonal antibodies against TRIM56 purchased from Abcam (Cambridge, United Kingdom, catalog no. 10583), and rabbit polyclonal antibodies against IRF3 (catalog no. 4302S), p-IRF3 (catalog no. 4947S), STING (catalog no. 13647S), p-STING (catalog no. 50907S), and α-tubulin (catalog no. 2148S) were purchased from Cell Signaling Technology (United States).

Techniques: Ubiquitin Proteomics, Infection, Western Blot, Transfection, Immunoprecipitation

Journal: PLoS Pathogens

Article Title: JMJD6 negatively regulates cytosolic RNA induced antiviral signaling by recruiting RNF5 to promote activated IRF3 K48 ubiquitination

doi: 10.1371/journal.ppat.1009366

Figure Lengend Snippet: (A) JMJD6 inhibits virus-induced IFN-β production at the IRF3 level. HEK293T cells were transfected with the indicated plasmids for 24 h. Luciferase assays were performed with a dual-specific luciferase assay kit. Protein expression was analyzed by Western blotting. (B) Endogenous JMJD6 reduces phosphorylated IRF3 (p-IRF3) and total IRF3. KO-control or KO-JMJD6 cell line was infected with or without SeV for 12 h. The p-IRF3, total IRF3, JMJD6, and β-actin were detected. (C) IRF3 is phosphorylated in the IRF3 overexpression situation. HEK293T cells were transfected with the indicated plasmids, and the expression of phosphorylated IRF3 was determined by immunoblot.

Article Snippet: Antibodies against p-IRF3 (Cell Signaling Technology (CST), Cat #4947), IRF3 (CST, Cat #4302), p-TBK1 (CST, Cat #5483), TBK1 (CST, Cat #3013), Ubiquitin (CST, Cat #3936), Ubiquitin K63-specific linkage (CST, Cat #5621), Ubiquitin K48-specific linkage (CST, Cat #8081), HA (Biolegend, Cat #901513), Flag (Santa Cruz Biotechnology, Cat #sc-166355), Myc (Santa Cruz Biotechnology, Cat #sc-47694), and β-actin (Santa Cruz Biotechnology, Cat #sc-47778), JMJD6 (Abcom, Cat #ab176172 and Proteintech, Cat #16476-1-AP), and GAPDH (Proteintech, Cat #60004-1-Ig) were purchased from the indicated manufacturers.

Techniques: Virus, Transfection, Luciferase, Expressing, Western Blot, Control, Infection, Over Expression

Journal: PLoS Pathogens

Article Title: JMJD6 negatively regulates cytosolic RNA induced antiviral signaling by recruiting RNF5 to promote activated IRF3 K48 ubiquitination

doi: 10.1371/journal.ppat.1009366

Figure Lengend Snippet: (A) HEK293T cells and HEK293T-JMJD6-Flag cell lines were infected with SeV as indicated, and the phosphorylation levels of IRF3 and TBK1 were analyzed. (B) Diagrams of IRF3 and its mutants. DBD, DNA binding domain; IAD, IRF3 association domain; RD, a C-terminal regulatory domain (left panel). HEK293T cells were transfected with the indicated plasmids, and cell lysates were immunoprecipitated with anti-Myc or IgG antibody followed by immunoblot using anti-HA and anti-Myc antibodies (right panel). (C) Endogenous JMJD6 interacts with IRF3 or p-IRF3 after a viral infection or poly(I:C) stimulation. HEK293T cells were infected with SeV for 12 h. Cells lysates were immunoprecipitated with anti-IRF3 or anti-p-IRF3, and the immunoprecipitates were analyzed by immunoblot with anti-JMJD6 antibody (top panel). Immunoblot of lysates from HEK293T cells stimulated with poly(I:C) for 12 h, analyzed with anti-IRF3 antibody (below panel). (D) Diagrams of JMJD6 and its mutants (Left panel). HEK293T cells transfected with the indicated plasmids and stimulated with poly(I:C) as indicated. The cell lysates were immunoprecipitated with an IgG or anti-HA antibody followed by immunoblots using anti-HA and anti-Myc antibodies. Asterisks represent target proteins (Right panel). (E) HEK293T cells were co-transfected with vector, Myc-JMJD6 or the JMJD6 mutants expressing plasmids and the IFN-β promoter-reporter plasmids for 24 h, and then infected with or without SeV for another 12 h. The luciferase activity was measured with a dual-luciferase assay. Expression of Myc-tagged JMJD6 protein and the mutant proteins was evaluated by Western blotting. (F) HEK293T cells were transfected with the indicated plasmids for 24 h and then infected with SeV for another 12 h. The phosphorylated IRF3 (p-IRF3), total IRF3, Myc-JMJD6 or mutants, and β-actin were detected by Western blotting.

Article Snippet: Antibodies against p-IRF3 (Cell Signaling Technology (CST), Cat #4947), IRF3 (CST, Cat #4302), p-TBK1 (CST, Cat #5483), TBK1 (CST, Cat #3013), Ubiquitin (CST, Cat #3936), Ubiquitin K63-specific linkage (CST, Cat #5621), Ubiquitin K48-specific linkage (CST, Cat #8081), HA (Biolegend, Cat #901513), Flag (Santa Cruz Biotechnology, Cat #sc-166355), Myc (Santa Cruz Biotechnology, Cat #sc-47694), and β-actin (Santa Cruz Biotechnology, Cat #sc-47778), JMJD6 (Abcom, Cat #ab176172 and Proteintech, Cat #16476-1-AP), and GAPDH (Proteintech, Cat #60004-1-Ig) were purchased from the indicated manufacturers.

Techniques: Infection, Phospho-proteomics, Binding Assay, Transfection, Immunoprecipitation, Western Blot, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Mutagenesis

Journal: PLoS Pathogens

Article Title: JMJD6 negatively regulates cytosolic RNA induced antiviral signaling by recruiting RNF5 to promote activated IRF3 K48 ubiquitination

doi: 10.1371/journal.ppat.1009366

Figure Lengend Snippet: (A) Endogenous JMJD6 regulates the stability of IRF3. KO-control cells, KO-JMJD6 cells, or KO-JMJD6 cells reconstituted with JMJD6 were infected with SeV for 6 h and then cultured in the presence of cycloheximide (CHX) for 0, 6, 12 h. Total IRF3, Myc-JMJD6, and β-actin were detected. (B) HEK293T cells were transfected with the indicated plasmids and then stimulated with poly(I:C) in the presence or absence of MG132 (20 μM), CQ (100 μM), or Z-VAD-FMK (50 μM) for 6 h. The expression of HA-IRF3 and Myc-JMJD6 proteins were detected by Western blotting. (C) Endogenous JMJD6 enhanced the ubiquitination of IRF3. KO-control cells, KO-JMJD6 cells, or KO-JMJD6 cells reconstituted with JMJD6 were infected with SeV for 6 h in the presence of MG132 (20 μM). Immunoblot analysis (with anti-Ub) of proteins immunoprecipitated (with anti-IRF3) was performed.

Article Snippet: Antibodies against p-IRF3 (Cell Signaling Technology (CST), Cat #4947), IRF3 (CST, Cat #4302), p-TBK1 (CST, Cat #5483), TBK1 (CST, Cat #3013), Ubiquitin (CST, Cat #3936), Ubiquitin K63-specific linkage (CST, Cat #5621), Ubiquitin K48-specific linkage (CST, Cat #8081), HA (Biolegend, Cat #901513), Flag (Santa Cruz Biotechnology, Cat #sc-166355), Myc (Santa Cruz Biotechnology, Cat #sc-47694), and β-actin (Santa Cruz Biotechnology, Cat #sc-47778), JMJD6 (Abcom, Cat #ab176172 and Proteintech, Cat #16476-1-AP), and GAPDH (Proteintech, Cat #60004-1-Ig) were purchased from the indicated manufacturers.

Techniques: Control, Infection, Cell Culture, Transfection, Expressing, Western Blot, Ubiquitin Proteomics, Immunoprecipitation

Journal: PLoS Pathogens

Article Title: JMJD6 negatively regulates cytosolic RNA induced antiviral signaling by recruiting RNF5 to promote activated IRF3 K48 ubiquitination

doi: 10.1371/journal.ppat.1009366

Figure Lengend Snippet: (A) List of JMJD6 interactome based on Label-free quantification intensity. (B) Immunoblot analysis (with anti-Myc or anti-Flag antibodies) of proteins immunoprecipitated (with control IgG or anti-Flag) from lysates of HEK293T cells transfected with the indicated plasmids. The expression of the transfected proteins was analyzed by immunoblot. (C) HEK293T cells were transfected with the indicated plasmids, followed by immunoprecipitation with an IgG or anti-Flag antibody and immunoblotting with anti-Flag or anti-Myc antibodies. (D) HEK293T cells were transfected with the indicated plasmids, followed by immunoprecipitation with an IgG or anti-Myc antibody and immunoblotting with anti-Flag or anti-Myc antibodies. (E) Immunoblot analysis (with anti-Ub) of proteins immunoprecipitated (with anti-HA) from lysates of HEK293T cells transfected for 24 h with various combinations of plasmids (upper panels) and stimulated with poly(I:C) in the presence of MG132. The expression of Flag-RNF5, Myc-JMJD6, or HA-IRF3 was determined by immunoblot with the indicated antibodies (lower panels). (F) Immunoblot analysis (with anti-ubiquitin or antibody to K63-linked or K48-linked polyubiquitin (K63-ub or K48-ub, respectively); top) of proteins immunoprecipitated (with anti-HA) from lysates of HEK293T cells transfected for 24 h with various combinations of plasmids (upper panels) and stimulated with poly(I:C) in the presence of MG132. The expression of Flag-RNF5, Flag-RNF5C42S, Myc-JMJD6, or HA-IRF3 was determined by immunoblot with the indicated antibodies (lower panels). (G) Immunoblot analysis of RNF5 KO efficiency in HEK293T cells (Left panel). Immunoblot analysis of RNF5 KO cells transfected with Myc-JMJD6 or vector and secondarily transfected with poly(I:C), analyzed with anti-IRF3, anti-RNF5, and anti-Myc antibodies (Middle panel). The rescue experiment by ectopically expressing full-length of RNF5 in RNF5-knockout cells (Right panel). (H) In vitro interaction analysis of JMJD6 with RNF5 or IRF3 using in vitro-translated JMJD6, RNF5, and IRF3. JMJD6, RNF5, and IRF3 were obtained by in vitro transcription and translation. Far-left panel, the interaction between JMJD6 and RNF5 or IRF3 was assayed by mixing JMJD6 and RNF5 or IRF3, followed by IP with JMJD6 antibody. Left panel, the interaction between JMJD6 and IRF3, followed by IP with JMJD6 antibody. Right panel, the interaction between JMJD6 and RNF5, followed by IP with JMJD6 antibody. Far-right panel, the interaction between IRF3 and RNF5, followed by IP with IRF3 antibody. (I) Immunoblot analysis of both IRF3 and RNF5 in a JMJD6-immunoprecipitated complex upon virus infection. HEK293T cells were transfected for 24 h with or without Myc-JMJD6 in the presence of SeV infection. (J) Fluorescent images of HEK293T cells stimulated with or without SeV (MOI of 1) for 12 h. DAPI-stained nuclei are shown in blue. Left panel, RNF5 was detected with green fluorescence. Right panel, JMJD6 was detected with red fluorescence, and IRF3 was detected with green fluorescence.

Article Snippet: Antibodies against p-IRF3 (Cell Signaling Technology (CST), Cat #4947), IRF3 (CST, Cat #4302), p-TBK1 (CST, Cat #5483), TBK1 (CST, Cat #3013), Ubiquitin (CST, Cat #3936), Ubiquitin K63-specific linkage (CST, Cat #5621), Ubiquitin K48-specific linkage (CST, Cat #8081), HA (Biolegend, Cat #901513), Flag (Santa Cruz Biotechnology, Cat #sc-166355), Myc (Santa Cruz Biotechnology, Cat #sc-47694), and β-actin (Santa Cruz Biotechnology, Cat #sc-47778), JMJD6 (Abcom, Cat #ab176172 and Proteintech, Cat #16476-1-AP), and GAPDH (Proteintech, Cat #60004-1-Ig) were purchased from the indicated manufacturers.

Techniques: Quantitative Proteomics, Western Blot, Immunoprecipitation, Control, Transfection, Expressing, Ubiquitin Proteomics, Plasmid Preparation, Knock-Out, In Vitro, Virus, Infection, Staining, Fluorescence

Journal: PLoS Pathogens

Article Title: JMJD6 negatively regulates cytosolic RNA induced antiviral signaling by recruiting RNF5 to promote activated IRF3 K48 ubiquitination

doi: 10.1371/journal.ppat.1009366

Figure Lengend Snippet: JMJD6 suppresses the RLR sensing pathway by degrading activated IRF3 in an RNF5-dependent manner.

Article Snippet: Antibodies against p-IRF3 (Cell Signaling Technology (CST), Cat #4947), IRF3 (CST, Cat #4302), p-TBK1 (CST, Cat #5483), TBK1 (CST, Cat #3013), Ubiquitin (CST, Cat #3936), Ubiquitin K63-specific linkage (CST, Cat #5621), Ubiquitin K48-specific linkage (CST, Cat #8081), HA (Biolegend, Cat #901513), Flag (Santa Cruz Biotechnology, Cat #sc-166355), Myc (Santa Cruz Biotechnology, Cat #sc-47694), and β-actin (Santa Cruz Biotechnology, Cat #sc-47778), JMJD6 (Abcom, Cat #ab176172 and Proteintech, Cat #16476-1-AP), and GAPDH (Proteintech, Cat #60004-1-Ig) were purchased from the indicated manufacturers.

Techniques:

Journal: PLOS Pathogens

Article Title: Basal IFNλ2/3 signaling is required for ISG expression and viral control in human intestinal epithelial cells

doi: 10.1371/journal.ppat.1013857

Figure Lengend Snippet: (A) qRT-PCR analysis of select ISGs Mx1, OAS1, ISG15, IRF7, RIG-I, and IFIT1 in T84 WT, IFNλ1 KO, IFNλ2/3 KO, and IFNLR KO three days post-seeding. Relative expression was normalized to TBP. (B) Western blot analysis of select ISGs (Mx1, IRF7, RIG-I, ISG15 and STAT1) in T84 WT, IFNλ1 KO, IFNλ2/3 KO, and IFNLR KO three days post-seeding. Mx1, IRF7, RIG-I, ISG15 and STAT1 protein abundance was quantified relative to actin as loading control. Representative images shown. (C) T84 WT, IFNλ1 KO, IFNλ2/3 KO cells were treated with recombinant IFNl1-3 proteins (100ng/mL) and cells were collected at 0-, 1-, 3-, and 6-hours post-treatment. Western Blot analysis of p-STAT1 and STAT1 was performed. P-STAT1 and STAT1 abundances were quantified relative to actin as loading control. Representative images shown. (D) Same as (C) but ISG (Mx1, OAS1, ISG15 and IFIT1) induction was assessed by qRT-PCR 24 h post-treatment. Relative expression was normalized to TBP. Data represent n ≥ 3 biological replicates. Statistical significance was determined using two-way ANOVA (*P < 0.05, P < 0.01 **, P < 0.001 ***, P < 0.0001 ****, ns = not significant). Error bars represent standard deviation with the mean as the center.

Article Snippet: Membranes were incubated with primary antibodies against IRF3 (Cell Signaling #11904T), Mx1 (Santa Cruz #sc-271024), IRF7 (Cell Signaling Technologies # 5184S), RIG-I (AdipoGen # AG-20B-0009), ISG15 (Santa Cruz #166755), STAT1 (BD Biosciences #610115), phospho-STAT1 (BD Biosciences #612233), and actin (Sigma Aldrich #A5441) diluted 1:1000 in blocking buffer overnight at 4°C.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Quantitative Proteomics, Control, Recombinant, Standard Deviation

Journal: PLOS Pathogens

Article Title: Basal IFNλ2/3 signaling is required for ISG expression and viral control in human intestinal epithelial cells

doi: 10.1371/journal.ppat.1013857

Figure Lengend Snippet: (A–C) T84 WT, IFNλ1 KO, IFNλ2/3 KO, and IRF3 KO cells were seeded in 6 well plates as 2x10 6 cells/well, and the media was changed the following day with 1.5 mL fresh media. Two days later, the cell supernatant was collected after centrifugation at 2000rpm for 5 minutes (referred as conditioned media), and used to treat T84 WT and IFNLR KO cells. Cells were treated with culture media (DMEM-F12) as control. (A) Schematic representation of experimental design was created in BioRender Keser,Y. (2025) https://BioRender.com/6ln3qq4 . (B) At 1-hour post-treatment (hpt), cells were harvested for Western blot analysis of STAT1 phosphorylation. P-STAT1 protein abundance was quantified relative to total actin, loading control. Representative images shown.(C) At 24 hours post-treatment, cells were harvested to assess ISG induction. qRT-PCR analysis of ISGs (Mx1, IFIT1, and ISG15) was performed following treatment by conditioned media. Relative expression was normalized to TBP. Data represent n ≥ 3 biological replicates. Statistical significance was determined using two-way ANOVA (*P < 0.05, P < 0.01 **, P < 0.001 ***, P < 0.0001 ****, ns = not significant). Error bars represent standard deviation with the mean as the center.

Article Snippet: Membranes were incubated with primary antibodies against IRF3 (Cell Signaling #11904T), Mx1 (Santa Cruz #sc-271024), IRF7 (Cell Signaling Technologies # 5184S), RIG-I (AdipoGen # AG-20B-0009), ISG15 (Santa Cruz #166755), STAT1 (BD Biosciences #610115), phospho-STAT1 (BD Biosciences #612233), and actin (Sigma Aldrich #A5441) diluted 1:1000 in blocking buffer overnight at 4°C.

Techniques: Centrifugation, Control, Western Blot, Phospho-proteomics, Quantitative Proteomics, Quantitative RT-PCR, Expressing, Standard Deviation

Journal: PLOS Pathogens

Article Title: Basal IFNλ2/3 signaling is required for ISG expression and viral control in human intestinal epithelial cells

doi: 10.1371/journal.ppat.1013857

Figure Lengend Snippet: (A–F) T84 WT, IFNλ1 KO, IFNλ2/3 KO, and IRF3 KO cells were seeded in 6 well plates as 2x10 6 cells/well, and the media was replaced the following day with 1.5 mL fresh media. Two days later, the cell supernatant was collected after centrifugation at 2000rpm for 5 minutes (referred to as conditioned media), and used to treat T84 IRF3 KO cells for 24 hours. Cells treated with culture media (DMEM-F12) served as a control. At 24 h post-treatment, cells were infected. (A) Schematic representation of experimental design was created in BioRender Keser,Y. (2025) https://BioRender.com/f9bbe51 . (B, C) VSV-GFP, (D) VSV_Luc, and (E, F) RV-UnaG. (B) VSV-GFP infection was assessed by live-cell imaging at 7 hpi, with nuclei stained using Hoechst. (C) Quantification of B. (C) VSV-Luc replication was assessed by luciferase assay at 7 hpi. (D) RV-UnaG infection (16 hpi) was evaluated by live-cell imaging, with nuclei stained using Hoechst. (F) Quantification of E. (B, E) Representative images shown. Scale bar = 100 μm. Data represent n ≥ 3 biological replicates. Statistical significance was determined using two-way ANOVA ( P < 0.05 *, P < 0.01 **, P < 0.001 ***, P < 0.0001 ****, ns = not significant). Error bars represent standard deviation with the mean as the center.

Article Snippet: Membranes were incubated with primary antibodies against IRF3 (Cell Signaling #11904T), Mx1 (Santa Cruz #sc-271024), IRF7 (Cell Signaling Technologies # 5184S), RIG-I (AdipoGen # AG-20B-0009), ISG15 (Santa Cruz #166755), STAT1 (BD Biosciences #610115), phospho-STAT1 (BD Biosciences #612233), and actin (Sigma Aldrich #A5441) diluted 1:1000 in blocking buffer overnight at 4°C.

Techniques: Centrifugation, Control, Infection, Live Cell Imaging, Staining, Luciferase, Standard Deviation

Journal: PLOS Pathogens

Article Title: Basal IFNλ2/3 signaling is required for ISG expression and viral control in human intestinal epithelial cells

doi: 10.1371/journal.ppat.1013857

Figure Lengend Snippet: (A) Schematic of the conditioned-media (CM) neutralization workflow was created in BioRender Keser,Y. (2025) https://BioRender.com/drh0ch2 . T84 WT cells were seeded in 6 well plates as 2x10 6 cells/well, and the media was replaced the following day with 1.5 mL fresh media. Two days later, the cell supernatant was collected after centrifugation at 2000rpm for 5 minutes (referred to as conditioned media (CM)). This conditioned media were incubated with neutralizing antibodies targeting IFNλ1 (α-λ1), IFNλ2 (α-λ2), IFNλ3 (α-λ3), IFNλ2/3 (α-λ2/3), or all three subtypes (α-λ1/2/3) for 1 h at room temperature. Antibody-treated CM were applied to T84 WT cells for analysis of STAT1 phosphorylation (1 h post-treatment) and ISG expression (16 h post-treatment). (B) Representative Western blots showing pSTAT1, total STAT1, and actin as a loading control following treatment with antibody-depleted CM. p-STAT1 protein abundance was quantified relative to STAT1. (C) qRT-PCR analysis of MX1 expression (normalized to TBP) 16 h after antibody-depleted CM treatment. (D) Same as A except CM were used to pre-treat T84 IRF3-KO cells for 24 h prior to VSV-Luc (MOI = 1) infection to assess antiviral activity at 7 hpi. Created in BioRender Keser,Y. (2025) https://BioRender.com/1zuiu9o . (E) VSV-Luciferase assay in T84 IRF3-KO cells pre-treated with antibody-depleted CM at 7 hpi. Data represent n ≥ 3 biological replicates. Statistical significance was determined using one-way ANOVA with multiple-comparison correction (*P < 0.05, P < 0.01 **, P < 0.001 ***, P < 0.0001 ****, ns = not significant). Error bars represent standard deviation with the mean as the center.

Article Snippet: Membranes were incubated with primary antibodies against IRF3 (Cell Signaling #11904T), Mx1 (Santa Cruz #sc-271024), IRF7 (Cell Signaling Technologies # 5184S), RIG-I (AdipoGen # AG-20B-0009), ISG15 (Santa Cruz #166755), STAT1 (BD Biosciences #610115), phospho-STAT1 (BD Biosciences #612233), and actin (Sigma Aldrich #A5441) diluted 1:1000 in blocking buffer overnight at 4°C.

Techniques: Neutralization, Centrifugation, Incubation, Phospho-proteomics, Expressing, Western Blot, Control, Quantitative Proteomics, Quantitative RT-PCR, Infection, Activity Assay, Luciferase, Comparison, Standard Deviation

Journal: PLOS Pathogens

Article Title: Basal IFNλ2/3 signaling is required for ISG expression and viral control in human intestinal epithelial cells

doi: 10.1371/journal.ppat.1013857

Figure Lengend Snippet: (A, B) T84 WT cells were seeded, and media was replaced the following day. After 48 h, supernatants (conditioned media) were collected and used as a reference control for antiviral activity. IRF3 KO cells were treated with recombinant IFNλ2 or IFNλ3 (0.01–20 ng/mL) or with WT conditioned media for 24 h and then infected with VSV-Luc for 7 h. (A) Schematic representation of the experimental workflow was created in BioRender Keser,Y. (2025) https://BioRender.com/ip2l074 . (B) 7hpi luciferase activity was measured to assess VSV-Luc infection in IRF3 KO cells treated with recombinant IFNλ2 or IFNλ3. (C–F) IFNλ2/3 KO cells were chronically supplemented for two weeks with IFNλ2 (5 ng/mL), IFNλ3 (1 ng/mL), or both. Cells were then trypsinized, reseeded in the absence of any IFN treatment and collected 48 h later for ISG analysis, or used for antiviral assays. (C) Schematic representation of chronic IFNλ2/3 supplementation and subsequent experimental steps. Created in BioRender Keser,Y. (2025) https://BioRender.com/3775duy . (D) Western blot analysis of IRF7, RIG-I, and STAT1 in WT cells and IFNλ2/3 KO cells under the indicated supplementation conditions or non-treated (NT). Protein abundance was quantified relative to actin. Representative images are shown. (E) qRT-PCR analysis of ISGs (MX1, IFIT1, OAS1) in WT cells and IFNλ2/3 cells maintained with IFNλ2, IFNλ3, IFNλ2 + 3, or non-treated. Relative expression was normalized to TBP. (F) VSV-Luc infection was measured by luciferase assayed 7 hpi in hours in WT and IFNλ2/3 cells maintained with IFNλ2, IFNλ3, IFNλ2 + 3, or non-treated. (G–I) IFNλ2/3 KO cells were chronically supplemented with IFNλ2 (5 ng/mL), IFNλ3 (1 ng/mL), or IFNλ2 + 3 for two weeks, reseeded in the absence of any IFNs, and next day, acutely stimulated with IFNλ1–3 (20 ng/mL of each) for 1 h or 24 h. (G) Schematic representation of chronic supplementation followed by acute IFNλ stimulation, was created in BioRender Keser,Y. (2025) https://BioRender.com/beodbxz . (H) Western blot analysis of p-STAT1 and total STAT1 in WT and IFNλ2/3 cells maintained with IFNλ2, IFNλ3, IFNλ2 + 3, or non-treated (NT). Protein abundance was quantified relative to actin, loading control. Representative images are shown. (I) qRT-PCR analysis of ISGs (MX1, IFIT1, OAS1) 24 h after acute IFNλ1–3 stimulation in WT and ΔIFNλ2/3 cells supplemented as indicated. Relative expression was normalized to TBP. Data represent n ≥ 3 biological replicates. Statistical significance was determined using two-way ANOVA (P < 0.05 *, P < 0.01 **, P < 0.001 ***, P < 0.0001 ****, ns = not significant). Error bars represent standard deviation, with the mean shown at the center.

Article Snippet: Membranes were incubated with primary antibodies against IRF3 (Cell Signaling #11904T), Mx1 (Santa Cruz #sc-271024), IRF7 (Cell Signaling Technologies # 5184S), RIG-I (AdipoGen # AG-20B-0009), ISG15 (Santa Cruz #166755), STAT1 (BD Biosciences #610115), phospho-STAT1 (BD Biosciences #612233), and actin (Sigma Aldrich #A5441) diluted 1:1000 in blocking buffer overnight at 4°C.

Techniques: Control, Activity Assay, Recombinant, Infection, Luciferase, Western Blot, Quantitative Proteomics, Quantitative RT-PCR, Expressing, Standard Deviation

Journal: PLOS Pathogens

Article Title: Basal IFNλ2/3 signaling is required for ISG expression and viral control in human intestinal epithelial cells

doi: 10.1371/journal.ppat.1013857

Figure Lengend Snippet: In WT cells (left panel), both IFNλ1 and IFNλ2/3 are produced under homeostatic conditions via IRF3 activation. Secreted IFNλs engage the IFNLR receptor on neighboring cells, activating the JAK/STAT pathway and inducing robust expression of ISGs thereby limiting viral replication. In IFNλ1 KO cells (middle panel), IFNλ2/3 are still expressed and can activate STAT1/2 signaling and ISG expression, maintaining effective antiviral defense with only a minor reduction in ISG levels. In contrast, IFNλ2/3 KO cells (right panel) retain IFNλ1 expression but exhibit a dramatic loss of STAT1/2 expression and fail to activate ISG transcription, resulting in impaired JAK/STAT signaling and increased viral replication. These findings highlight the predominant and non-redundant role of IFNλ2/3 in establishing and sustaining the basal antiviral state in intestinal epithelial cells. Schematics were created in BioRender Keser,Y. (2025) https://BioRender.com/3oi4mf0 .

Article Snippet: Membranes were incubated with primary antibodies against IRF3 (Cell Signaling #11904T), Mx1 (Santa Cruz #sc-271024), IRF7 (Cell Signaling Technologies # 5184S), RIG-I (AdipoGen # AG-20B-0009), ISG15 (Santa Cruz #166755), STAT1 (BD Biosciences #610115), phospho-STAT1 (BD Biosciences #612233), and actin (Sigma Aldrich #A5441) diluted 1:1000 in blocking buffer overnight at 4°C.

Techniques: Produced, Activation Assay, Expressing