Journal: Journal of Nanobiotechnology

Article Title: Apoptotic tumor cell-derived microparticles loading Napabucasin inhibit CSCs and synergistic immune therapy

doi: 10.1186/s12951-023-01792-8

Figure Lengend Snippet: In vivo validation of the mechanism of NAP inhibiting colon cancer. A The KEGG enrichment analysis indicated that the immune-related pathway was regulated after TMPs treatment. B GSEA algorithm was used to analyze the changes in the cytosolic DNA-sensing pathway after TMPs treatment. C p-TBK1(Ser172) and p-IRF3(Ser386), markers of activation of innate immune pathway cytosolic DNA-sensing, were detected after TMPs treatment. D – F RT-qPCR detected downstream target genes of the DNA-sensing pathway. G – H Representative flow analysis of CD3 + CD4 + T cells and CD3 + CD8 + T cells in tumor tissues. I – J Representative fluorescent images of CD3 + CD4 + T cells and CD3 + CD8 + T cells in tumor tissues. (n = 6, scale bars:100 μm)

Article Snippet: GAPDH antibody (#ab8245, 1:3000) was purchased from Abcam. p-TBK1(Ser172) antibody (#5483,1:1000), p-IRF3(Ser386) (#37829, 1:1000) were purchased from Cell Signaling Technology.

Techniques: In Vivo, Activation Assay, Quantitative RT-PCR

Journal: Journal of Medicinal Chemistry

Article Title: New Glucosamine-Based TLR4 Agonists: Design, Synthesis, Mechanism of Action, and In Vivo Activity as Vaccine Adjuvants

doi: 10.1021/acs.jmedchem.2c01998

Figure Lengend Snippet: Immunofluorescence analysis of p-IRF-3 nuclear translocation at 2 h. Phospho-IRF-3 localization in THP-1-derived macrophages (TDM) after LPS stimulation and FP20 25 μM treatment at t = 2 h.

Article Snippet: Subsequently, cells were labeled with NF-κB p65 XP Rabbit mAb (1:400; 8242, Cell Signaling Technology, Inc.) or Phospho-IRF-3 (Ser386) XP Rabbit mAb (1:400; E7J8G, Cell Signaling Technology, Inc.).

Techniques: Immunofluorescence, Translocation Assay, Derivative Assay

Journal: Journal of Medicinal Chemistry

Article Title: New Glucosamine-Based TLR4 Agonists: Design, Synthesis, Mechanism of Action, and In Vivo Activity as Vaccine Adjuvants

doi: 10.1021/acs.jmedchem.2c01998

Figure Lengend Snippet: Immunofluorescence analysis of p-IRF-3 nuclear translocation at 4 h. Phospho-IRF-3 localization in THP-1-derived macrophages (TDM) after LPS stimulation and FP20 25 μM treatment at t = 4 h.

Article Snippet: Subsequently, cells were labeled with NF-κB p65 XP Rabbit mAb (1:400; 8242, Cell Signaling Technology, Inc.) or Phospho-IRF-3 (Ser386) XP Rabbit mAb (1:400; E7J8G, Cell Signaling Technology, Inc.).

Techniques: Immunofluorescence, Translocation Assay, Derivative Assay

Journal: Cell Death and Differentiation

Article Title: CD-NTase family member MB21D2 promotes cGAS-mediated antiviral and antitumor immunity

doi: 10.1038/s41418-023-01116-1

Figure Lengend Snippet: A Western blot analysis of p-STING, p-TBK1 and p-IRF3 in WT and Mb21d2 -/- peritoneal macrophages after ISD or cGAMP stimulation, or HSV-1 infection for indicated times. B Western blot analysis of p-TBK1 and p-IRF3 in sg Ctrl, sg KO, and sg KO reintroduced of MB21D2 HeLa cells after ISD or cGAMP stimulation, or HSV-1 infection for indicated times. C Microscopy images of IRF3 nuclear translocation in sg Ctrl or sg KO HeLa cells after ISD stimulation for 4 h (left). The percentage of cells with nuclear translocation was quantified (right), at least 100 cells from each group were analyzed, n = 3. Scale bar, 20 μm. D Microscopy images of IRF3 nuclear translocation in WT or Mb21d2 -/- MEFs after ISD stimulation for 4 h (left). The percentage of cells with nuclear translocation was quantified (right), at least 100 cells from each group were analyzed, n = 3. Scale bar, 20 μm. Data are shown as mean ± SD and were analyzed by unpaired two-tailed Student’s t test ( C , D ) (* p < 0.05, ** p < 0.01).

Article Snippet: The following antibodies were used: anti-cGAS (#31659), anti-cGAS (#83623), anti-TBK1 (#3013), anti-phospho-TBK1 (#5483), anti-IRF3 (#4302), anti- phospho-IRF3 (#4947), anti-Myc (#2276) were purchased from Cell Signaling Technology; anti-VSV-G (V5507), anti-MB21D2 (SAB2108874) were purchased from Sigma-Aldrich; anti-Flag (PA1-984B) was purchased from Thermo Fisher Scientific; anti-HA (sc-7392), anti-Actin (sc-58673) were purchased from Santa Cruz Biotechnology; FITC anti-mouse CD3 (#100203), PE anti-mouse IFN-γ (#505807), APC anti-mouse Perforin (#154403), PE/Cy7-anti-mouse CD80 (#104733), PerpCP/Cyanine5.5 anti-mouse CD86 (#105027), FITC anti-mouse CD11c (#117305) were purchased from BioLegend; PE anti-mouse CD62L (#561918), PerCP-Cy5.5 anti-mouse CD8α (#561109), APC anti-mouse CD69 (#560689), PE/Cyanine7 anti-human/mouse Granzyme B (#372213) were purchased from BD Biosciences; APC anti-mouse-MHC class II (ab93559) was purchased from Abcam.

Techniques: Western Blot, Infection, Microscopy, Translocation Assay, Two Tailed Test

Journal: Cell Death and Differentiation

Article Title: CD-NTase family member MB21D2 promotes cGAS-mediated antiviral and antitumor immunity

doi: 10.1038/s41418-023-01116-1

Figure Lengend Snippet: A HEK293T cells were respectively transfected with Myc-tagged cGAS, STING, RIG-I, MAVS, TBK1, or IRF3 together with Flag-tagged MB21D2 for 24 h. Coimmunoprecipitations and immunoblots were performed with indicated antibodies. B Coimmunoprecipitations and immunoblots of MB21D2 with cGAS in THP-1 cells after ISD stimulation for indicated times. C Microscopy images of cGAS and MB21D2 colocalization in HeLa cells. Pearson correlation coefficient (PCC) was analyzed by Image J. Scale bar, 5 μm. D Coimmunoprecipitations and immunoblots of purified His-tagged hcGAS FL or hcGAS-ΔN Mutant protein with purified MB21D2 protein. E Coimmunoprecipitations and immunoblots of purified His-tagged mcGAS FL or mcGAS-ΔN Mutant protein with purified MB21D2 protein. F Coimmunoprecipitations and immunoblots of purified His-tagged MB21D2 FL or MB21D2 Mutant protein with purified mcGAS protein.

Article Snippet: The following antibodies were used: anti-cGAS (#31659), anti-cGAS (#83623), anti-TBK1 (#3013), anti-phospho-TBK1 (#5483), anti-IRF3 (#4302), anti- phospho-IRF3 (#4947), anti-Myc (#2276) were purchased from Cell Signaling Technology; anti-VSV-G (V5507), anti-MB21D2 (SAB2108874) were purchased from Sigma-Aldrich; anti-Flag (PA1-984B) was purchased from Thermo Fisher Scientific; anti-HA (sc-7392), anti-Actin (sc-58673) were purchased from Santa Cruz Biotechnology; FITC anti-mouse CD3 (#100203), PE anti-mouse IFN-γ (#505807), APC anti-mouse Perforin (#154403), PE/Cy7-anti-mouse CD80 (#104733), PerpCP/Cyanine5.5 anti-mouse CD86 (#105027), FITC anti-mouse CD11c (#117305) were purchased from BioLegend; PE anti-mouse CD62L (#561918), PerCP-Cy5.5 anti-mouse CD8α (#561109), APC anti-mouse CD69 (#560689), PE/Cyanine7 anti-human/mouse Granzyme B (#372213) were purchased from BD Biosciences; APC anti-mouse-MHC class II (ab93559) was purchased from Abcam.

Techniques: Transfection, Western Blot, Microscopy, Purification, Mutagenesis

Journal: Frontiers in Immunology

Article Title: CRISPR-mediated rapid arming of poxvirus vectors enables facile generation of the novel immunotherapeutic STINGPOX

doi: 10.3389/fimmu.2022.1050250

Figure Lengend Snippet: STINGPOX induces IFN signaling in a STING-dependent manner. (A) Primary murine BMDCs were infected with STINGPOX or OncoVACV for 24 h. Whole cell lysates were analyzed by WB for phospho-STING (p-STING S365), STING, phospho-TBK1 (p-TBK1 S172), TBK1, phospho-IRF3 (p-IRF3 S396), IRF3, IFIT1, cGAS and FLAG (for disA expression). β-actin levels were used as loading control. Data are representative of three independent experiments. (B) THP1-Dual cells were infected with STINGPOX or OncoVACV (at a range of MOIs) and relative luciferase activity was assessed as a measure of IFN signaling (n = 3). (C) Supernatants IFN-β levels in THP1-Dual infected with STINGPOX or OncoVACV (MOI of 0.3 or 1) for 24 h were measured by ELISA. (D) Heatmap of relative fold change in expression (to mock) in primary human macrophages infected with OncoVACV or STINGPOX (MOI = 3) for 18 hours, and analyzed by RNA-seq. Log2 transformed data is shown for genes associated Jak/STAT or IFN αβ signaling pathways that were activated at least 4-fold in STINGPOX infected cells relative to OncoVACV infection conditions. (E) IFN signaling measured as described in (B) using WT, cGAS KO, and STING KO THP1-Dual cells. (F) Immunoblot analyses of primary murine WT and cGAS -/- BMDCs infected with STINGPOX or OncoVACV, as described in (A) . Data are shown as mean ± SEM.

Article Snippet: The primary antibodies used for Western blotting were from the following sources: Abcam: IFIT1 (ab11821); Cell Signaling Technology: phospho-TBK1 (S172, #5483), TBK1 (3504), phospho-IRF3 (S396, #4947), IRF3 (#4302), phospho-STING (S365, #72971), STING (#3337), FLAG (#14793), cGAS (#31659); Sigma: β-actin antibody (A5441).

Techniques: Infection, Expressing, Luciferase, Activity Assay, Enzyme-linked Immunosorbent Assay, RNA Sequencing Assay, Transformation Assay, Western Blot

Journal: Frontiers in Microbiology

Article Title: RIG-I and MDA5 are modulated by bone morphogenetic protein (BMP6) and are essential for restricting Zika virus infection in human Sertoli cells

doi: 10.3389/fmicb.2022.1062499

Figure Lengend Snippet: Viral RNA-sensing and type I IFN-associated pathways are functional in SC. (A) SC were infected with ZIKV at MOI 1 and ZIKV titers in the supernatant were measured at different time points using plaque assay and expressed as PFU/mL supernatant. (B) Cell viability was evaluated in infected SC at different days after infection using CellTiter 96 AQueous One Solution kit and shown as percent cell viability compared to mock-infected cells. (C) SC were infected with ZIKV at MOI 1 and gene expression of IFN-β , MDA5 , RIG-I , TLR3 , TLR7 , and TLR9 were measured using qRT-PCR at 48 h post-infection (hpi). The fold change was calculated as compared to mock-infected cells and normalized to GAPDH . (D) Whole-cell extracts from mock and infected SC at 48 hpi were subjected to Western blotting and stained for IRF3, p-IRF3, and MXA. The β-actin was used as a housekeeping loading control and each lane represents an independent experiment. (E) SC were pretreated with poly (I:C) 24 h before infection at MOI 1 and ZIKV RNA copies were determined at 48 hpi by qRT-PCR and expressed as ZIKV PFU equivalents per μg RNA. (F) SC and A549 cells were pretreated with exogenous IFN-β at two concentrations 24 h before infection and ZIKV titers in the supernatant were measured at 48 hpi using plaque assay and expressed as PFU/mL. Data is presented as mean ± SEM from at least 3 to 5 independent experiments. ** p < 0.01 between respective IFN-β treated and untreated cells.

Article Snippet: Specific primary monoclonal antibodies used were rabbit anti-human against RIG-I (Cell Signaling Cat. No: 3743S), MDA5 (Invitrogen Cat. No: 700360), MXA (Santa Cruz Biotechnology Cat. No: sc-271024), IRF3 and phospho-IRF3 (Cell Signaling Cat. No: 11904S and Cat. No: 4947S), STAT1 (Cell Signaling Cat. No: 9172S), phospho-STAT1 (Cell Signaling Cat. No: 9167L), STAT2 (Cell Signaling Cat. No: 72604S), phospho-STAT2 (Cell Signaling Cat. No 4441) and mouse anti-human β-actin (Sigma Cat. No. A2228-200UL), all at 1:1,000 dilution.

Techniques: Functional Assay, Infection, Plaque Assay, Expressing, Quantitative RT-PCR, Western Blot, Staining

Journal: Frontiers in Microbiology

Article Title: RIG-I and MDA5 are modulated by bone morphogenetic protein (BMP6) and are essential for restricting Zika virus infection in human Sertoli cells

doi: 10.3389/fmicb.2022.1062499

Figure Lengend Snippet: RIG-I restricts ZIKV replication in human SC. (A,B) SC were transfected with either siCtrl or siRNA specific to exon 3 or exon 11 of RIG-I. The silencing efficiency was determined by measuring RIG-I protein levels using Western blot at 48 hpt transfection. (C) SC were infected with ZIKV (MOI 1) at 48 hpt and ZIKV titers was quantified using plaque assay at different time points. (D) Wild-type (WT) and RIG-I knock-out A549 were infected with ZIKV (MOI 1) and virus titers in the supernatants were determined by plaque assay at 48 hpi and expressed as PFU/mL supernatant. (E) Whole-cell extracts from uninfected and infected SC transfected with siCtrl, and siRIG-I (exon 11) at 48 hpi with ZIKV were subjected to Western blotting and stained for p-IRF3 and β-actin. (F) Densitometry analysis was performed using ImageJ and the p-IRF3/β-actin ratio in infected cells relative to the siCtrl mock was calculated. Data is presented as mean ± SEM from at least 3–5 independent experiments. * p < 0.05; ** p < 0.01.

Article Snippet: Specific primary monoclonal antibodies used were rabbit anti-human against RIG-I (Cell Signaling Cat. No: 3743S), MDA5 (Invitrogen Cat. No: 700360), MXA (Santa Cruz Biotechnology Cat. No: sc-271024), IRF3 and phospho-IRF3 (Cell Signaling Cat. No: 11904S and Cat. No: 4947S), STAT1 (Cell Signaling Cat. No: 9172S), phospho-STAT1 (Cell Signaling Cat. No: 9167L), STAT2 (Cell Signaling Cat. No: 72604S), phospho-STAT2 (Cell Signaling Cat. No 4441) and mouse anti-human β-actin (Sigma Cat. No. A2228-200UL), all at 1:1,000 dilution.

Techniques: Transfection, Western Blot, Infection, Plaque Assay, Knock-Out, Staining

Journal: Frontiers in Microbiology

Article Title: RIG-I and MDA5 are modulated by bone morphogenetic protein (BMP6) and are essential for restricting Zika virus infection in human Sertoli cells

doi: 10.3389/fmicb.2022.1062499

Figure Lengend Snippet: Silencing of MDA5 increases ZIKV replication in SC. (A) SC were transfected with siRNA specific for siMDA5 or siCtrl and silencing efficiency was determined by measuring MDA5 protein levels using Western blot at 48 hpt. (B) Percent change in the cell viability of siCtrl and siMDA5 transfected SC at 48 hpt was calculated as compared to untreated cells. (C) SC were infected with ZIKV (MOI 1) at 48 hpt and ZIKV PFU were determined by plaque assay at different time points, expressed as PFU/mL supernatant. (D) WT and MDA5 knockout A549 were infected with ZIKV (MOI 1) and virus titers in the supernatants were determined by plaque assay at 48 hpi. (E) The effect of siMDA5 on phosphorylated IRF3 (p-IRF3) protein expression was determined in both mock-and ZIKV-infected SC at 48 hpi by Western blotting. (F) Densitometry analysis was performed to quantify western blot data and expressed as the ratio of p-IRF3/β-actin in infected cells relative to the siCtrl mock. (G) SC were co-transfected with siMDA5, and siRIG-1 (exon 11), and knockdown was determined by measuring mRNA transcripts. (H) ZIKV titers in the supernatants from siCtrl and double knock-down (DKD) with siMDA5 and siRIG-I cells were determined by plaque assay at 48 hpi and expressed as PFU/mL supernatant. (I) Percent increase in ZIKV titers in SC transfected with siRIG-I and siMDA5 compared to siCtrl at 48 hpi. (J) Gene expression of IFNB1, MXA, and IFIT1 was determined at 48 hpi using qRT-PCR. Data was normalized using GAPDH and fold change was calculated compared to respective mock-infected cells. Data represent mean ± SEM from at least 3–5 independent experiments. * p < 0.05; ** p < 0.01.

Article Snippet: Specific primary monoclonal antibodies used were rabbit anti-human against RIG-I (Cell Signaling Cat. No: 3743S), MDA5 (Invitrogen Cat. No: 700360), MXA (Santa Cruz Biotechnology Cat. No: sc-271024), IRF3 and phospho-IRF3 (Cell Signaling Cat. No: 11904S and Cat. No: 4947S), STAT1 (Cell Signaling Cat. No: 9172S), phospho-STAT1 (Cell Signaling Cat. No: 9167L), STAT2 (Cell Signaling Cat. No: 72604S), phospho-STAT2 (Cell Signaling Cat. No 4441) and mouse anti-human β-actin (Sigma Cat. No. A2228-200UL), all at 1:1,000 dilution.

Techniques: Transfection, Western Blot, Infection, Plaque Assay, Knock-Out, Expressing, Quantitative RT-PCR

Journal: Frontiers in Microbiology

Article Title: RIG-I and MDA5 are modulated by bone morphogenetic protein (BMP6) and are essential for restricting Zika virus infection in human Sertoli cells

doi: 10.3389/fmicb.2022.1062499

Figure Lengend Snippet: BMP6 enhances the antiviral response in SC. (A) BMP6 mRNA transcripts were measured in SC, A549, and BMVEC at 24 h post-treatment with 1 IU/mL of recombinant human IFN-β using RT-PCR. (B–D) SC were treated with recombinant human IFN-β in the presence and absence of recombinant BMP6 (100 ng/mL) for 24 h and mRNA transcripts of MXA, IFIT1, IRF7 , and STAT1 were measured by qRT-PCR. SC treated with poly (I:C) in the presence and absence of BMP6 and mRNA expression of (E) IFNB1 , and (G) IFIT1, and MXA was measured by qRT-PCR. (F) IFN-β levels in the supernatant from treated SC were measured by ELISA and expressed as pg/mL supernatant. (H) Whole-cell extracts from IFN-β and poly I:C exposed SC in the presence and absence of BMP6 were subjected to Western blotting and stained for p-IRF3, total and p-STAT1, total and p-STAT2, and β-actin. Data is presented as mean ± SEM from at least 3–5 independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Specific primary monoclonal antibodies used were rabbit anti-human against RIG-I (Cell Signaling Cat. No: 3743S), MDA5 (Invitrogen Cat. No: 700360), MXA (Santa Cruz Biotechnology Cat. No: sc-271024), IRF3 and phospho-IRF3 (Cell Signaling Cat. No: 11904S and Cat. No: 4947S), STAT1 (Cell Signaling Cat. No: 9172S), phospho-STAT1 (Cell Signaling Cat. No: 9167L), STAT2 (Cell Signaling Cat. No: 72604S), phospho-STAT2 (Cell Signaling Cat. No 4441) and mouse anti-human β-actin (Sigma Cat. No. A2228-200UL), all at 1:1,000 dilution.

Techniques: Recombinant, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Staining