Journal: Cellular & molecular immunology

Article Title: The zinc-finger protein ZFYVE1 modulates TLR3-mediated signaling by facilitating TLR3 ligand binding.

doi: 10.1038/s41423-019-0265-6

Figure Lengend Snippet: Fig. 2 ZFYVE1 is important for TLR3-mediated signaling. a Effects of ZFYVE1-RNAi on the expression of ZFYVE1. As shown in the upper two panels, HEK293 cells (1 × 105) were transfected with HA-ZFYVE1 (0.1 μg), HA-β-actin (0.01 μg), and the indicated ZFYVE1-RNAi plasmids (0.5 μg) for 24 h before immunoblotting analysis. As shown in the lower two panels, HEK293 cells (1 × 105) were transfected with the indicated ZFYVE1-RNAi plasmids (0.5 μg) for 12 h. The cells were then selected with puromycin (1 μg/mL) for 24 h before immunoblotting analysis. b Effects of ZFYVE1-RNAi on poly(I:C)-induced activation of the IFN-β promoter, ISRE, and NF-κB. 293-TLR3 cells (1 × 105) were transfected with the indicated reporter and ZFYVE1-RNAi plasmids (0.5 μg) for 36 h and then were or were not treated with poly(I:C) (20 μg/mL) for 6 h before luciferase assays. c Effects of ZFYVE1-RNAi on the poly(I:C)-induced transcription of downstream genes. 293-TLR3 cells (1 × 105) were transfected with control or ZFYVE1-RNAi plasmid (1 μg) for 12 h. The cells were selected with puromycin (1 μg/mL) for 24 h and then were or were not treated with poly(I:C) (50 μg/mL) for 3 h before qPCR analysis. d Effects of ZFYVE1 deficiency on the poly(I:C)-induced transcription of downstream genes. ZFYVE1-deficient HT1080 cells were generated by the CRISPR-Cas9 method. ZFYVE1-KO and control HT1080 cells (1 × 105) were or were not treated with poly(I:C) (50 μg/mL) for the indicated times before qPCR analysis. e Effects of ZFYVE1 deficiency on the LPS- induced transcription of downstream genes. ZFYVE1-deficient 293-TLR4 cells were generated by the CRISPR-Cas9 method. ZFYVE1-KO and control 293-TLR4 cells (1 × 105) were or were not treated with LPS (100 ng/mL) for 2 h before qPCR analysis. f Effects of ZFYVE1 deficiency on the poly(I:C)- and LPS-induced phosphorylation of TBK1, IRF3, and p65. ZFYVE1-KO and control cells (1 × 105) were or were not treated with poly(I:C) (50 μg/mL) or LPS (100 ng/mL) for the indicated times before immunoblotting analysis. Data are represented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student’s t test)

Article Snippet: Reagents, antibodies, and cells The following reagents, antibodies, and cells were purchased from the indicated companies: TRIzol (TaKaRa Bio); SYBR Green (BioRad); a dual-specific luciferase assay kit (Promega); polybrene (Millipore); poly(I:C), PGN, and R848 (Invivogen); LPS and DNase I (Sigma); EZ-link Psoralen-PEG3-Biotin (Thermo), type II collagenase (Worthington), a first strand cDNA synthesis kit (Fermentas), GammaBind G Plus-Sepharose (Amersham Biosciences), a cell mitochondria isolation kit (Beyotime), a nuclear and cytoplasmic Cellular & Molecular Immunology _#####################_ extraction kit (Applygen); ELISA kits to detect murine IFN-β, TNFα, and IL-6 (BioLegend); mouse monoclonal antibodies against Flag, β-actin (Sigma), HA (Origene), p-IRF3 (Cell Signaling Technology), p-p65 (Cell Signaling Technology), TLR3 (Cell Signaling Technology), p-Tyr (Cell Signaling Technology), IRF3 (Santa Cruz Biotechnology), AIF (Santa Cruz Biotechnology), KDEL (Santa Cruz Biotechnology), p65 (Abcam), TBK1 (Abcam), p-TBK1 (Abcam), TRIF (Abcam), LMNB1 (Proteintech), β-tubulin (Life Technology), ZFYVE1 and RAB5A (Abclonal); and Alexa Fluor 488- and Alexa Fluor 594-conjugated goat anti-mouse IgG antibodies (Invitrogen).

Techniques: Expressing, Transfection, Western Blot, Activation Assay, Luciferase, Control, Plasmid Preparation, Generated, CRISPR, Phospho-proteomics

Journal: Cellular & molecular immunology

Article Title: The zinc-finger protein ZFYVE1 modulates TLR3-mediated signaling by facilitating TLR3 ligand binding.

doi: 10.1038/s41423-019-0265-6

Figure Lengend Snippet: Fig. 3 Zfyve1 deficiency attenuates TLR3-mediated signaling in primary mouse cells. a Effects of Zfyve1 deficiency on the poly(I:C)- and LPS- induced transcription of downstream genes. Zfyve1+/+ and Zfyve1−/−MLFs (2 × 105) were or were not treated with poly(I:C) (50 μg/mL) or LPS (100 ng/mL) for the indicated times before qPCR analysis. b Effects of Zfyve1 deficiency on the poly(I:C)- and LPS-induced transcription of downstream genes. Zfyve1+/+ and Zfyve1−/−BMDCs (2 × 105) were or were not treated with poly(I:C) (20 μg/mL) or LPS (100 ng/mL) for the indicated times before qPCR analysis. c Effects of Zfyve1 deficiency on the PGN- or R848-induced transcription of downstream genes. Zfyve1+/+ and Zfyve1−/−MLFs or BMDCs (2 × 105) were or were not treated with PGN (20 mg/mL) or R848 (10 mM) for 3 h before qPCR analysis. d Effects of Zfyve1 deficiency on the poly(I:C)-induced phosphorylation of IRF3 and p65. Zfyve1+/+ and Zfyve1−/−BMDCs (2 × 105) were or were not treated with poly(I:C) (50 μg/mL) or LPS (100 ng/mL) for the indicated times before immunoblotting analysis. e Effects of Zfyve1 deficiency on the transcription of downstream genes induced by different doses of poly(I:C). Zfyve1+/+ and Zfyve1−/−MLFs (2 × 105) were or were not treated with increasing amounts of poly(I:C) for 3 h before qPCR analysis. The percentage reduction in the transcription of the Ifnb1 and Isg56 genes in Zfyve1−/−MLFs compared with that in Zfyve1+/+ MLFs is shown in the right panels. Data are represented as the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 (Student’s t test)

Article Snippet: Reagents, antibodies, and cells The following reagents, antibodies, and cells were purchased from the indicated companies: TRIzol (TaKaRa Bio); SYBR Green (BioRad); a dual-specific luciferase assay kit (Promega); polybrene (Millipore); poly(I:C), PGN, and R848 (Invivogen); LPS and DNase I (Sigma); EZ-link Psoralen-PEG3-Biotin (Thermo), type II collagenase (Worthington), a first strand cDNA synthesis kit (Fermentas), GammaBind G Plus-Sepharose (Amersham Biosciences), a cell mitochondria isolation kit (Beyotime), a nuclear and cytoplasmic Cellular & Molecular Immunology _#####################_ extraction kit (Applygen); ELISA kits to detect murine IFN-β, TNFα, and IL-6 (BioLegend); mouse monoclonal antibodies against Flag, β-actin (Sigma), HA (Origene), p-IRF3 (Cell Signaling Technology), p-p65 (Cell Signaling Technology), TLR3 (Cell Signaling Technology), p-Tyr (Cell Signaling Technology), IRF3 (Santa Cruz Biotechnology), AIF (Santa Cruz Biotechnology), KDEL (Santa Cruz Biotechnology), p65 (Abcam), TBK1 (Abcam), p-TBK1 (Abcam), TRIF (Abcam), LMNB1 (Proteintech), β-tubulin (Life Technology), ZFYVE1 and RAB5A (Abclonal); and Alexa Fluor 488- and Alexa Fluor 594-conjugated goat anti-mouse IgG antibodies (Invitrogen).

Techniques: Phospho-proteomics, Western Blot

Journal: BMC Veterinary Research

Article Title: Curcumol inhibits encephalomyocarditis virus by promoting IFN-β secretion

doi: 10.1186/s12917-021-03015-4

Figure Lengend Snippet: Curcumol (0.025, 0.0125 and 0.00625 mg/mL) and ribavirin (0.25, 0.125 and 0.0625 mg/mL) were selected to treat EMCV-infected HEK-293 T cells for 24 h, and the expression of MDA5, MAVS, TANK, IRF3, P-IRF3 protein were detected by Western blot. Densitometric values of protein bands were quantified by the Image J. Data were analyzed using GraphPad Prism™ software 5.0 (GraphPad Software, Inc. California, USA). One-way analysis of variance (ANOVA) followed by a Dunnett’s post-test was used to determine the difference between the groups. All groups are compared with EMCV-infected group (* P < 0.05, ** P < 0.01, *** P < 0.001)

Article Snippet: MDA5 and MAVS Rabbit Polyclonal antibody, GAPDH Mouse Monoclonal antibody, Goat anti-Mouse and Goat anti-Rabbit secondary antibodies were purchased from Proteintech Biotechnology Co., Ltd. (China), TANK Rabbit Polyclonal antibody and IRF3 Rabbit Monoclonal antibody from Abcam (USA); and Phospho-IRF3 Ribbit Polyclonal antibody from Bioss (China).

Techniques: Infection, Expressing, Western Blot, Software

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

Article Title: Endoplasmic reticulum stress regulates the innate immunity critical transcription factor interferon regulatory factor 3

doi: 10.4049/jimmunol.1102737

Figure Lengend Snippet: Oxygen glucose deprivation (OGD) synergistically augments LPS-induced IFN-β and causes IRF3 nuclear translocation. A) MEFs were subjected to 0, 0.5, 1, or 2 h OGD, followed by media with, or without 10ng/mL LPS for 3h in standard culture conditions. IFN-β expression was determined by quantitative PCR (qPCR) with normalization to 18S rRNA (Relative mRNA). Bars represent combined results from 3 independent experiments with error bars showing SEM. p<0.0005 comparing OGD pretreated conditions and no OGD. Comparable results have been obtained in 5 independent experiments. B) MEFs were subjected to 1h OGD followed by standard culture conditions as above. Expression of BiP and spliced XBP1 was detected by qPCR and normalized to untreated controls for fold induction (NT=1). Results are representative of 2 individual experiments and error bars denote S.D. P<0.0004 comparing OGD treated and NT cells. C) MEFs were subjected to OGD for 1h followed by 2h re-oxygenation (Re-ox) in standard culture as indicated. Fixed cells were incubated with anti-IRF3 followed by anti-rabbit IgG Alexa Fluor 488, and visualized by immunofluorescence microscopy. Results are representative of 3 independent experiments. D) Nuclear and cytoplasmic lysates were resolved by SDS PAGE and immunoblotted with anti-IRF3, β-actin, or the nuclear protein anti-USF2. Results are representative of 2 separate experiments.

Article Snippet: β-actin, mouse mAb, NF-κB rabbit polyclonal Ab, and USF2 rabbit polyclonal Ab were from Santa Cruz; HA-Tag mouse mAb, IRF3 rabbit mAb (detects 1 IRF3 band) and phospho-IRF3 (pS396) rabbit mAb from Cell Signaling; Phospho-IRF3 (pS386) rabbit mAb from Epitomics; IRF3 rabbit polyclonal (detects 2 IRF3 forms), STING rabbit polyclonal Ab, and ATF6 mAb from Prosci; TBK1 rabbit mAb from Abcam; normal rabbit IgG from Millipore.

Techniques: Translocation Assay, Expressing, Real-time Polymerase Chain Reaction, Incubation, Immunofluorescence, Microscopy, SDS Page

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

Article Title: Endoplasmic reticulum stress regulates the innate immunity critical transcription factor interferon regulatory factor 3

doi: 10.4049/jimmunol.1102737

Figure Lengend Snippet: ER stress induces IRF3 nuclear translocation and phosphorylation. A) MEFs were treated with 1 μM Tg for the times indicated. Nuclear lysates were resolved by SDS page and immunoblotted with anti-IRF3 or β-actin control. Results are representative of 4 independent experiments. B) MEFs were treated with 1 μM Tg for 1 or 2h, fixed, incubated with anti-IRF3 followed by anti-rabbit IgG Alexa Fluor 488, and visualized by immunofluorescence microscopy. Results are representative of 3 independent experiments. C) XBP1-/- MEFs were treated with Tg for 0, 60 or 90 min and western blots incubated with anti-IRF3 or anti USF2. Results are representative of 3 independent experiments. D) MEFs were left untreated or subjected to OGD for 1h, 10μg/mL tunicamycin (Tm) 6h, 20mM 2-deoxyglucose (2DG) 6h, 1 μM Tg for 2h, or LPS 10ng/mL for 2h. Fixed cells were incubated with anti-pS386 IRF3 followed by anti-rabbit IgG Alexa Fluor 488, and visualized by immunofluorescence microscopy. Results are representative of 4 independent experiments. E) Wild type (WT) or IRF3-/- primary bone marrow derived macrophages were treated with 1μM Tg for 2h prior to fixation and staining for p-IRF3 (Ser 386). Results are representative of 2 independent experiments.

Article Snippet: β-actin, mouse mAb, NF-κB rabbit polyclonal Ab, and USF2 rabbit polyclonal Ab were from Santa Cruz; HA-Tag mouse mAb, IRF3 rabbit mAb (detects 1 IRF3 band) and phospho-IRF3 (pS396) rabbit mAb from Cell Signaling; Phospho-IRF3 (pS386) rabbit mAb from Epitomics; IRF3 rabbit polyclonal (detects 2 IRF3 forms), STING rabbit polyclonal Ab, and ATF6 mAb from Prosci; TBK1 rabbit mAb from Abcam; normal rabbit IgG from Millipore.

Techniques: Translocation Assay, Phospho-proteomics, SDS Page, Control, Incubation, Immunofluorescence, Microscopy, Western Blot, Derivative Assay, Staining

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

Article Title: Endoplasmic reticulum stress regulates the innate immunity critical transcription factor interferon regulatory factor 3

doi: 10.4049/jimmunol.1102737

Figure Lengend Snippet: ER stress augments LPS induction of some, but not all IRF3 regulated genes. A) Bone marrow derived macrophages were treated with 1μM Tg, 10ng/ml LPS or 1μM Tg+ 10ng/ml LPS for 3 hours. Relative expression of IFN-β, IFN-α4, RANTES, and ifit2 was determined by qPCR with normalization to 18S rRNA. Bars depict combined means of 6 (IFN-β, *p=0.001), 3 (IFN-α4, *p<0.006), and 4 (RANTES and ifit2) independent experiments with error bars showing the SEM. To combine IFN-α4, RANTES and ifit2 experiments, relative expression was normalized (fold induction vs. LPS=1). B) Macrophages were treated as above for 8h and supernatants assessed for cytokine and chemokine protein by ELISA. Bars represent standard error of triplicate determinations. *P<0.00002 (IFN-β).

Article Snippet: β-actin, mouse mAb, NF-κB rabbit polyclonal Ab, and USF2 rabbit polyclonal Ab were from Santa Cruz; HA-Tag mouse mAb, IRF3 rabbit mAb (detects 1 IRF3 band) and phospho-IRF3 (pS396) rabbit mAb from Cell Signaling; Phospho-IRF3 (pS386) rabbit mAb from Epitomics; IRF3 rabbit polyclonal (detects 2 IRF3 forms), STING rabbit polyclonal Ab, and ATF6 mAb from Prosci; TBK1 rabbit mAb from Abcam; normal rabbit IgG from Millipore.

Techniques: Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay

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

Article Title: Endoplasmic reticulum stress regulates the innate immunity critical transcription factor interferon regulatory factor 3

doi: 10.4049/jimmunol.1102737

Figure Lengend Snippet: Thapsigargin induces co-localization of TBK1 and STING and requires STING and TBK1 family kinases for IRF3 phosphorylation. A) MEFs were pre-treated with 2μM MRT67307 for 30 min prior to 1μM Tg for 2h. Fixed cells were incubated with anti-pIRF3 (pS386) followed by Alexafluor488 coupled secondary antibody and immunofluorescence detected by microscopy. Results are representative of 4 independent experiments. B) MEFs were untreated or treated with 1μM Tg for 2h. Samples were immunoprecipitated with control IgG or anti-STING. Whole cell lysate (input) or immunoprecipitations were resolved by SDS-PAGE and immunoblotted with anti-TBK1 or anti-STING. Results are representative of 4 independent experiments. C) MEFs were transfected with STING/MPYS-HA 24h prior to stimulation with 1μM TPG for 2h. Fixed cells were incubated with rabbit anti-TBK1 and mouse anti-HA followed by anti-rabbit Alexa Fluor 488 (green) or anti-mouse Alexa Fluor 594 (red) secondary antibodies. Results are representative of 3 independent experiments. D) WT or STING-/- MEFs were treated with 1 μM Tg for 2h as indicated. Cells were then fixed, incubated with anti-pS386 IRF3 followed by anti-rabbit IgG Alexa Fluor 488, and visualized by immunofluorescence microscopy. Results are representative of 4 independent experiments. E) WT and STING-/- MEFs were stimulated with 1μM Tg, 10ng/mL LPS, or 1μM Tg+10ng/ml LPS for 2 hrs. Nuclear lysates were resolved by SDS-PAGE and immunoblotted with antibodies specific for pIRF3 (S396), total IRF3, NF-κB, or USF2. Results are representative of 3 independent experiments.

Article Snippet: β-actin, mouse mAb, NF-κB rabbit polyclonal Ab, and USF2 rabbit polyclonal Ab were from Santa Cruz; HA-Tag mouse mAb, IRF3 rabbit mAb (detects 1 IRF3 band) and phospho-IRF3 (pS396) rabbit mAb from Cell Signaling; Phospho-IRF3 (pS386) rabbit mAb from Epitomics; IRF3 rabbit polyclonal (detects 2 IRF3 forms), STING rabbit polyclonal Ab, and ATF6 mAb from Prosci; TBK1 rabbit mAb from Abcam; normal rabbit IgG from Millipore.

Techniques: Phospho-proteomics, Incubation, Immunofluorescence, Microscopy, Immunoprecipitation, Control, SDS Page, Transfection

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

Article Title: Endoplasmic reticulum stress regulates the innate immunity critical transcription factor interferon regulatory factor 3

doi: 10.4049/jimmunol.1102737

Figure Lengend Snippet: STING-dependent IRF3 phosphorylation requires both calcium mobilization and ER stress. A) WT or STING-/- MEFs were pre-treated with 2μM MRT67307 for 30 min then 10μg/mL Tm for 6h as indicated. Cells were incubated with anti-pIRF3 (S386) followed by Alexa Fluor 488 secondary antibody and visualized by immunofluorescence microscopy. Results are representative of 4 independent experiments. B) WT or STING-/- MEFs were untreated (NT), treated with 1μM ionomycin or 2μM A23187 as indicated. Fixed cells were incubated with anti-pIRF3 followed by Alexa Fluor 488 secondary antibody and visualized by immunofluorescence microscopy. Results are representative of 3 independent experiments. C) Wild type or STING-/- MEFs were subjected to OGD (as in Figure 1) for 1h followed by 2 hours re-oxygenation. Cells were fixed (1C) or lysed (1D) and IRF3 nuclear translocation detected by immunofluorescence or western blot, respectively.

Article Snippet: β-actin, mouse mAb, NF-κB rabbit polyclonal Ab, and USF2 rabbit polyclonal Ab were from Santa Cruz; HA-Tag mouse mAb, IRF3 rabbit mAb (detects 1 IRF3 band) and phospho-IRF3 (pS396) rabbit mAb from Cell Signaling; Phospho-IRF3 (pS386) rabbit mAb from Epitomics; IRF3 rabbit polyclonal (detects 2 IRF3 forms), STING rabbit polyclonal Ab, and ATF6 mAb from Prosci; TBK1 rabbit mAb from Abcam; normal rabbit IgG from Millipore.

Techniques: Phospho-proteomics, Incubation, Immunofluorescence, Microscopy, Translocation Assay, Western Blot

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

Article Title: Endoplasmic reticulum stress regulates the innate immunity critical transcription factor interferon regulatory factor 3

doi: 10.4049/jimmunol.1102737

Figure Lengend Snippet: AEBSF, a site 1 protease inhibitor, blocks tunicamycin and 2-deoxyglucose induced IRF3 phosphorylation and tunicamycin-dependent synergistic IFN-β induction. A) RAW cells were pre-treated with 300 μM AEBSF for 1h, then stimulated with 1μM Tg for 2h, 20mM 2DG for 5h, or 10 μg/mL Tm for 5h. Cells were fixed and then stained with anti-pIRF3 (S386) plus secondary anti-rabbit Alexa Fluor488. Results are representative of 2 independent experiments. B) RAW cells were pretreated with AEBSF as in (A) and then untreated (NT), stimulated with Tg 1h, or Tm 5h, followed by an additional 3h media or LPS as indicated. Whole cell lysates were resolved by SDS PAGE and immunoblotted with anti-pIRF3 (S396), IRF-3 or actin. Results are representative of 2 independent experiments. C) RAW cells were pretreated with AEBSF as in (A), then stimulated with 1h Tg or 5h Tm followed by an additional 3h LPS as indicated. Relative IFN-β mRNA was quantified by qPCR with normalization to 18S rRNA. Results were combined from 4-5 independent experiments and error bars represent the standard error of the mean. * P<0.007. D) Cells were stimulated as in (B). Whole cell lysates were resolved by SDS PAGE and immunoblotted for precursor (P) and mature (M) cleaved forms of ATF6. Results are representative of 3 independent experiments.

Article Snippet: β-actin, mouse mAb, NF-κB rabbit polyclonal Ab, and USF2 rabbit polyclonal Ab were from Santa Cruz; HA-Tag mouse mAb, IRF3 rabbit mAb (detects 1 IRF3 band) and phospho-IRF3 (pS396) rabbit mAb from Cell Signaling; Phospho-IRF3 (pS386) rabbit mAb from Epitomics; IRF3 rabbit polyclonal (detects 2 IRF3 forms), STING rabbit polyclonal Ab, and ATF6 mAb from Prosci; TBK1 rabbit mAb from Abcam; normal rabbit IgG from Millipore.

Techniques: Protease Inhibitor, Phospho-proteomics, Staining, SDS Page