Journal: Cell Insight

Article Title: IRF1 amplifies HSV-1-triggered antiviral innate immunity in a feed-forward manner

doi: 10.1016/j.cellin.2025.100255

Figure Lengend Snippet: MITA activation is sufficient to induce IRF1 . (A) HT1080 cells were stimulated with diABZI (2.5 μM), and the expression of IRF1 , IFNB1 , ISG56, and CXCL10 was quantified by RT-qPCR at 6 h post-stimulation. (B) HT1080 cells were stimulated with diABZI (2.5 μM), and WCLs were analyzed by immunoblotting at 6 h post-stimulation. (C) THP-1 cells were stimulated with diABZI (2.5 μM), and the expression of IRF1 , IFNB1 , ISG56, and CXCL10 was quantified by RT-qPCR at 6 h post-stimulation. (D) THP-1 cells were stimulated with diABZI (2.5 μM), and WCLs were analyzed by immunoblotting at the indicated time points post-stimulation. (E) HT1080 cells were stimulated with MSA-2 (20 μM) or SR-717 (10 μM), and the expression of IRF1 , IFNB1 , ISG56, and CXCL10 was quantified by RT-qPCR at 8 h post-stimulation. (F) HT1080 cells were stimulated with MSA-2 (20 μM) or SR-717 (10 μM), and WCLs were analyzed by immunoblotting at 8 h post-stimulation.

Article Snippet: The chemical reagents used in this study include puromycin (InvivoGen), diABZI (Selleck), H-151 (MedChemExpress), MSA-2 (MedChemExpress) and SR-717 (MedChemExpress).

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: STING-induced noncanonical autophagy regulates endolysosomal homeostasis.

doi: 10.1073/pnas.2415422122

Figure Lengend Snippet: Fig. 1. STING activation elevates pH in Golgi-derived vesicles, leading to LC3 lipidation through V-ATPase and ATG16L1. (A) HeLa-STING(WT), HeLa-STING- ATG16L1KO, HeLa-STING-ATG16L1, and HeLa-STING-ATG16ΔWD40 cells were stimulated with 30 µM MSA2 for 2 h, or 100 nM Torin1 for 5 h, or untreated. The cell lysates were analyzed by immunoblotting as indicated. (B) HEK293T FIP200-KO cells stably expressing STING and GFP-LC3B were stimulated with indicated compounds (30 µM MSA2, 100 nM bafilomycin A1) for 2 h, or untreated. Cells were analyzed by confocal microscopy. (Scale bar, 10 µm.) (C) HeLa-STING-ATG16L1 and HeLa-STING-ATG16ΔWD40 cells were stimulated with 30 µM MSA2 for 1.5 h or untreated. Protein complexes were immunoprecipitated with a Flag antibody and analyzed by immunoblotting. (D) HeLa cells stably expressing STING, miRFP670-LC3B, and Super Ecliptic pHluorin (SEP)-mKate2-TGN46 were treated with 1 µM diABZI, and the cells were analyzed by live cell microscopy. Representative images taken at 0 min, 18 min, and 27 min are shown. The Left panels show the SEP signal of SEP-mKate2-TGN46 fusion protein. (Scale bar, 10 µm) for images on the Upper panels and 1 µm for Zoom-in images on the Bottom panels.

Article Snippet: The catalog numbers and vendors for chemicals are shown as the following: MSA2 (Tocris, 7353), diABZI (Cayman, 34082), C53 (Cayman,37354), Torin1 (Tocris, 296970), LLOMe (Bachem, 4000725.0001), bafilomycin A1 (Invivogen, tlrl- baf1), BFA (Invivogen, inh- bfa), GCA (EMD Millipore, 345862), Lysotracker Red (Invitrogen L7528), and pHrodo Red Dextran(P10361).

Techniques: Activation Assay, Derivative Assay, Western Blot, Stable Transfection, Expressing, Confocal Microscopy, Immunoprecipitation, Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: STING-induced noncanonical autophagy regulates endolysosomal homeostasis.

doi: 10.1073/pnas.2415422122

Figure Lengend Snippet: Fig. 2. Noncanonical autophagy is required for STING-induced activation of MiT/TFE transcription factors. (A) HeLa-STING (ctrl) and HeLa-STING-KO cells were treated with 30 µM MSA2 or 50 nM diABZI for 2 h or untreated, followed by analyses of cell lysates by immunoblotting with the indicated antibodies. (B) HeLa- STING cells stably expressing TFEB-miRFP670 were treated with 50 nM diABZI for 2 h or untreated, and live cells were imaged by microscopy. (Scale bar, 10 µm.) (C) Quantification of 77 and 80 cells per group as represented by B. Error bars represent SD. Tested by unpaired Student’s t test. ****P < 0.0001. (D) HeLa-STING, HeLa-STING-ATG16L1KO, HeLa-STING-ATG16L1, and HeLa-STING-ATG16ΔWD40 cells were stimulated with 30 µM MSA2 for 2 h or 100 nM Torin1 for 5 h or untreated. The cell lysates were analyzed by immunoblotting as indicated. (E) HeLa cells stably expressing STING (ctrl) or both STING and SopF (SopF) were treated with 30 µM MSA2 for 2 h or untreated. The cell lysates were analyzed by immunoblotting. (F) HeLa-STING, HeLa-STING-SopF, and HeLa-STING-ATG16L1KO cells stably expressing SEP-LC3B were treated with 30 µM MSA2 for 2 h or untreated. The cells were stained with an antibody against endogenous TFEB followed by confocal microscopy analyses. (Scale bar, 10 µm.) (G) Quantification of 88 to 228 cells per group as represented by F. Error bars represent SD. Tested by Tukey’s multiple comparisons test. ****P < 0.0001.

Article Snippet: The catalog numbers and vendors for chemicals are shown as the following: MSA2 (Tocris, 7353), diABZI (Cayman, 34082), C53 (Cayman,37354), Torin1 (Tocris, 296970), LLOMe (Bachem, 4000725.0001), bafilomycin A1 (Invivogen, tlrl- baf1), BFA (Invivogen, inh- bfa), GCA (EMD Millipore, 345862), Lysotracker Red (Invitrogen L7528), and pHrodo Red Dextran(P10361).

Techniques: Activation Assay, Western Blot, Stable Transfection, Expressing, Microscopy, Staining, Confocal Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: STING-induced noncanonical autophagy regulates endolysosomal homeostasis.

doi: 10.1073/pnas.2415422122

Figure Lengend Snippet: Fig. 3. TFEB activation by STING does not require TBK1. (A) HeLa-STING and HeLa-STING (1 to 370) cells were treated with 30 µM MSA2 for 2 h or untreated. Cell lysates were analyzed by immunoblotting. (B) HeLa-STING (1 to 370) cells were stimulated with 30 µM MSA2 for 2 h or untreated; then, cells were imaged by confocal microscopy using an antibody against TFEB. NucBlue stains nuclear DNA. (Scale bar, 10 µm.) (C) Quantification of 67 and 77 cells per group as represented by B. Error bars represent SD. Tested by unpaired Student’s t test. ****P < 0.0001. (D) HeLa-STING (1 to 370), and HeLa-STING (1 to 370)-SopF cells were treated with 10 µM MSA2 for 24 h or untreated. RNA was isolated from these cells for analysis by quantitative RT-PCR to measure the expression of indicated TFEB target genes. CTSD: cathepsin D; CTSB: cathepsin B; GPNMB: Glycoprotein Nonmetastatic Melanoma Protein B. The mRNA expression levels of the MSA-treated cells were normalized to untreated cells (ctrl). Error bars represent SD. The data are representative of 2 independent experiments. Tested by Tukey’s multiple comparisons test. ****P < 0.0001. (E) wild-type (WT) and TBK1/IKKε double knockout (DKO) THP1 cells were treated with 30 µM MSA2 for 2 h or untreated. Then cell lysates were analyzed by immunoblotting.

Article Snippet: The catalog numbers and vendors for chemicals are shown as the following: MSA2 (Tocris, 7353), diABZI (Cayman, 34082), C53 (Cayman,37354), Torin1 (Tocris, 296970), LLOMe (Bachem, 4000725.0001), bafilomycin A1 (Invivogen, tlrl- baf1), BFA (Invivogen, inh- bfa), GCA (EMD Millipore, 345862), Lysotracker Red (Invitrogen L7528), and pHrodo Red Dextran(P10361).

Techniques: Activation Assay, Western Blot, Confocal Microscopy, Isolation, Quantitative RT-PCR, Expressing, Double Knockout

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: STING-induced noncanonical autophagy regulates endolysosomal homeostasis.

doi: 10.1073/pnas.2415422122

Figure Lengend Snippet: Fig. 4. STING agonist C53 activates TBK1 and IRF3, but not TFEB, through a mechanism independent of membrane trafficking. (A) HeLa-STING cells were treated with 30 µM MSA2, 50 nM diABZI, or 10 µM C53 for 2 h or untreated, then cell lysates were analyzed by immunoblotting. (B) HeLa STING-KO cells stably expressing STING-mGFP and mCherry-Sec61B were treated with 1.25 µM C53, and the cells were analyzed by live cell microscopy. Representative images taken at 0 min (Ctrl) and 10 min (C53) are shown. (Scale bar, 10 µm) for images on the Left panels and 2 µm for Zoom-in images on the Right panels. (C) HeLa-STING cells were treated with 1 mM BFA or 10 mM GCA for 20 min before stimulation with 50 nM diABZI or 10 µM C53 for 2 h. Cell lysates were analyzed by immunoblotting with indicated antibodies. (D) HeLa-STING cells were stimulated with 1.25 µM C53 for 20 min or untreated, then cells were imaged by confocal microscopy using antibodies against STING and p-TBK1. NucBlue stains nuclear DNA. (Scale bar, 10 µm.)

Article Snippet: The catalog numbers and vendors for chemicals are shown as the following: MSA2 (Tocris, 7353), diABZI (Cayman, 34082), C53 (Cayman,37354), Torin1 (Tocris, 296970), LLOMe (Bachem, 4000725.0001), bafilomycin A1 (Invivogen, tlrl- baf1), BFA (Invivogen, inh- bfa), GCA (EMD Millipore, 345862), Lysotracker Red (Invitrogen L7528), and pHrodo Red Dextran(P10361).

Techniques: Membrane, Western Blot, Stable Transfection, Expressing, Microscopy, Confocal Microscopy

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: STING-induced noncanonical autophagy regulates endolysosomal homeostasis.

doi: 10.1073/pnas.2415422122

Figure Lengend Snippet: Fig. 5. FNIP1/FNIP2–GABARAP interactions mediate the activation of TFEB by STING. (A) HeLa-STING (Ctrl), HeLa-STING-FNIP1/2DKO, HeLa-STING-FNIP1WT, and HeLa-STING-FNIP1MUT cells were treated with 30 µM MSA2 for 2 h or untreated. Cell lysates were analyzed by immunoblotting as indicated. (B) HeLa-STING (WT), HeLa-STING-LC3sTKO, and HeLa-STING-GABARAPsTKO cells were treated with 30 µM MSA2 for 2 h or untreated. Cell lysates were analyzed by immunoblotting. (C) Similar to (B) except that cells were analyzed by immunofluorescent microscopy using an antibody against TFEB. TFEB+NucBlue: merging of TFEB staining with nuclear DNA staining by NucBlue. (Scale bar, 10 µm.) (D) Quantification of 46 to 76 cells per group as represented by C. Error bars represent SD. Tested by Tukey’s multiple comparisons test. ****P < 0.0001. (E) HeLa-STING cells were treated with 30 µM MSA2 or 50 nM diABZI for 2 h, or 100 nM Torin1 for 5 h, or untreated. Cell lysates were analyzed by immunoblotting. (F) Diagram depicting the mechanism by which STING-induced noncanonical autophagy promotes endolysosomal biogenesis through inhibiting the phosphorylation of TFEB, TFE3, and MITF by mTORC1.

Article Snippet: The catalog numbers and vendors for chemicals are shown as the following: MSA2 (Tocris, 7353), diABZI (Cayman, 34082), C53 (Cayman,37354), Torin1 (Tocris, 296970), LLOMe (Bachem, 4000725.0001), bafilomycin A1 (Invivogen, tlrl- baf1), BFA (Invivogen, inh- bfa), GCA (EMD Millipore, 345862), Lysotracker Red (Invitrogen L7528), and pHrodo Red Dextran(P10361).

Techniques: Activation Assay, Western Blot, Microscopy, Staining, Phospho-proteomics

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: STING-induced noncanonical autophagy regulates endolysosomal homeostasis.

doi: 10.1073/pnas.2415422122

Figure Lengend Snippet: Fig. 7. STING-induced autophagy recruits the ALIX–ESCRT to mitigate endolysosomal perturbation. (A) HeLa-STING, HeLa-STING-SopF, and HeLa-STING- ATG16L1KO cells that stably express SEP-LC3B were treated with 30 µM MSA2 for 2 h or untreated. Cells were imaged by microscopy using an antibody against ALIX and other markers as indicated. (Scale bar, 10 µm.) Arrows indicate ALIX puncta. (B) Quantification of 32 to 70 cells per group represented by A. Error bars represent SD. Tested by Tukey’s multiple comparisons test. ****P < 0.0001. (C) HeLa-STING cells transfected with siRNA against ALIX or control siRNA were treated with 50 nM diABZI for 24 h, and the morphology of the cells was analyzed by bright-field microscopy. (Scale bar, 25 µm.) (D) Quantification of 228 and 214 cells per group shown in C. Error bars represent SD. Tested by Tukey’s multiple comparisons test. ***P < 0.001. (E) HeLa-STING-ATG16L1 and HeLa-STING- ATG16ΔWD40 cells treated with 50 nM diABZI for 24 h or untreated, then cells were analyzed by bright-field microscopy. (Scale bar, 25 µm.) (F) Quantification of more than 300 cells per group shown in E. Error bars represent SD. Tested by unpaired Student’s t test. ***P < 0.001. (G) A Diagram showing that GABARAPs lipidation facilitates ALIX-mediated ESCRT recruitment which mitigates endolysosomal perturbation.

Article Snippet: The catalog numbers and vendors for chemicals are shown as the following: MSA2 (Tocris, 7353), diABZI (Cayman, 34082), C53 (Cayman,37354), Torin1 (Tocris, 296970), LLOMe (Bachem, 4000725.0001), bafilomycin A1 (Invivogen, tlrl- baf1), BFA (Invivogen, inh- bfa), GCA (EMD Millipore, 345862), Lysotracker Red (Invitrogen L7528), and pHrodo Red Dextran(P10361).

Techniques: Stable Transfection, Microscopy, Transfection, Control

Journal: Amino Acids

Article Title: Redefining an epitope of a malaria vaccine candidate, with antibodies against the N-terminal MSA-2 antigen of Plasmodium harboring non-natural peptide bonds

doi: 10.1007/s00726-013-1541-x

Figure Lengend Snippet: Schematic structure of the merozoite surface antigen-2. MSP-2 gene block organization is represented by different colors in regions and denoted by numbers from 1 to 5, highlighting the 4044 peptide NMR-solution structure located at the molecule N-terminal. Critical binding residues are underlined. MSA-2 is anchored to the parasite surface by a GPI motif

Article Snippet: Each level in the scale represents a sub-cloning of new antibody populations Fig. 7 Reactivity of Aotus polyclonal antibodies and hybridoma-IgM antibodies to MSA-2 peptide mimetics. a Antibody reactivity of hybridoma-IgM produced antibodies induced by MSA-2 peptide mimetics against a recombinant MSA-2 expressed in E. coli .

Techniques: Blocking Assay, Binding Assay

Journal: Amino Acids

Article Title: Redefining an epitope of a malaria vaccine candidate, with antibodies against the N-terminal MSA-2 antigen of Plasmodium harboring non-natural peptide bonds

doi: 10.1007/s00726-013-1541-x

Figure Lengend Snippet: Solid-phase synthesis of malaria MSA-2 antigen-derived peptide mimetics. ( 1 ) The amino primary function of Asn residue in the sequence anchored to the methylbenzhydrylamine resin (MBHA) was condensed with the carbonyl carbon of synthon t -Boc- L –Ile–H ( 2 ), thereby forming the iminium ion in the product ( 3 ). The formation of this ion led to a methylene group formation in product ( 4 ) after being treated with the NaCNBH 3 reducing agent. After incorporating the remaining normal amino acids into the peptide chain, the final expected product, ψ-130 ( 5 ), was generated when it was cleaved from the resin by treatment with low and high HF concentrations. SPPS was used for solid-phase peptide synthesis

Article Snippet: Each level in the scale represents a sub-cloning of new antibody populations Fig. 7 Reactivity of Aotus polyclonal antibodies and hybridoma-IgM antibodies to MSA-2 peptide mimetics. a Antibody reactivity of hybridoma-IgM produced antibodies induced by MSA-2 peptide mimetics against a recombinant MSA-2 expressed in E. coli .

Techniques: Derivative Assay, Sequencing, Generated

Journal: Amino Acids

Article Title: Redefining an epitope of a malaria vaccine candidate, with antibodies against the N-terminal MSA-2 antigen of Plasmodium harboring non-natural peptide bonds

doi: 10.1007/s00726-013-1541-x

Figure Lengend Snippet: The effect of introducing a single ψ–[CH 2 NH] peptide bond modification on the MSA-2 21–40 fragment backbone and its relevance to the molecule 3D structure. A consensus structure for peptide 4044 and its pseudopeptides ψ-128 and ψ-130, all having the lowest total energy are displayed in ball and sticks representing the molecular 3D structure of target molecules based on multidimensional 1 H-NMR experiments. The molecule backbone is shown by red ribbons. Conformation properties for peptide MSA-2 21−40 (peptide 4044) in panel ( a ) and its analog pseudopeptides ψ-128 in ( b ) and ψ-130 in ( c ). Left panels show all molecules’ side view and right panels show the molecules’ front view from the N- to C-terminus. Distance between 30 F and 38 S is shown in Å. Left panels show all molecules’ side view and right panels show the molecules’ front view from N- to C-terminus

Article Snippet: Each level in the scale represents a sub-cloning of new antibody populations Fig. 7 Reactivity of Aotus polyclonal antibodies and hybridoma-IgM antibodies to MSA-2 peptide mimetics. a Antibody reactivity of hybridoma-IgM produced antibodies induced by MSA-2 peptide mimetics against a recombinant MSA-2 expressed in E. coli .

Techniques: Modification

Journal: Amino Acids

Article Title: Redefining an epitope of a malaria vaccine candidate, with antibodies against the N-terminal MSA-2 antigen of Plasmodium harboring non-natural peptide bonds

doi: 10.1007/s00726-013-1541-x

Figure Lengend Snippet: Genealogical tree of antibody clones induced by pseudopeptides ψ-128 and ψ-130 derived from the Plasmodium falciparum MSA-2 antigen so-called HABP 4044. Each level in the scale represents a sub-cloning of new antibody populations

Article Snippet: Each level in the scale represents a sub-cloning of new antibody populations Fig. 7 Reactivity of Aotus polyclonal antibodies and hybridoma-IgM antibodies to MSA-2 peptide mimetics. a Antibody reactivity of hybridoma-IgM produced antibodies induced by MSA-2 peptide mimetics against a recombinant MSA-2 expressed in E. coli .

Techniques: Clone Assay, Derivative Assay, Subcloning

Journal: Amino Acids

Article Title: Redefining an epitope of a malaria vaccine candidate, with antibodies against the N-terminal MSA-2 antigen of Plasmodium harboring non-natural peptide bonds

doi: 10.1007/s00726-013-1541-x

Figure Lengend Snippet: Reactivity of Aotus polyclonal antibodies and hybridoma-IgM antibodies to MSA-2 peptide mimetics. a Antibody reactivity of hybridoma-IgM produced antibodies induced by MSA-2 peptide mimetics against a recombinant MSA-2 expressed in E. coli . Lane 1 for the negative control, lane 2 shows the reactivity of an anti-His antibody, lanes 3 and 4 show the reactivity of IgM induced by ψ-130 and lanes 5 and 6 the reactivity of IgM induced by ψ-128. b Antibody reactivity of Aotus monkeys immunized with MSA-2 peptide mimetics against a recombinant MSA-2 expressed in E. coli ; lane 1 the negative control, lane 2 the reactivity of an anti-His antibody, lane 3 the reactivity of polyclonal antibodies induced by ψ-130 in Aotus monkeys, lane 4 the reactivity of polyclonal antibodies induced by ψ-128 in Aotus monkeys, lanes 5 and 6 display the reactivity of pre-immune sera from monkeys and lanes 7 and 8 sera reactivity of pre-immune and post third dose samples of monkeys immunized with native 4044 peptide

Article Snippet: Each level in the scale represents a sub-cloning of new antibody populations Fig. 7 Reactivity of Aotus polyclonal antibodies and hybridoma-IgM antibodies to MSA-2 peptide mimetics. a Antibody reactivity of hybridoma-IgM produced antibodies induced by MSA-2 peptide mimetics against a recombinant MSA-2 expressed in E. coli .

Techniques: Produced, Recombinant, Negative Control

Journal: Amino Acids

Article Title: Redefining an epitope of a malaria vaccine candidate, with antibodies against the N-terminal MSA-2 antigen of Plasmodium harboring non-natural peptide bonds

doi: 10.1007/s00726-013-1541-x

Figure Lengend Snippet: Mapping the N-terminal MSA-2 epitope. The peptide 4044 glycine scan analog peptides is displayed in a , b . The reactivity pattern of monoclonal antibodies coded as G, M, O and Q induced by the pseudopeptide ψ-130 ( 30 F–I 31 ) against the 4044 peptide glycine scan. c . The reactivity pattern of monoclonal antibodies coded 6 and 7 induced by the pseudopeptide ψ-128 ( 31 I–N 32 ) against the 4044 peptide glycine scan. Each bar represents experiments performed in duplicate by ELISA. d Schematic representation of the MSA-2 21–40 N-terminal region in which a redefined epitope is shown in ( 3 ), which overlap the previously reported ( 1 ) (Jones et al. ) and a described HLA-DRβ1 reading frame motif ( 2 ) (Patarroyo et al. )

Article Snippet: Each level in the scale represents a sub-cloning of new antibody populations Fig. 7 Reactivity of Aotus polyclonal antibodies and hybridoma-IgM antibodies to MSA-2 peptide mimetics. a Antibody reactivity of hybridoma-IgM produced antibodies induced by MSA-2 peptide mimetics against a recombinant MSA-2 expressed in E. coli .

Techniques: Enzyme-linked Immunosorbent Assay