Journal: Frontiers in Immunology

Article Title: A Synthetic Small Molecule F240B Decreases NLRP3 Inflammasome Activation by Autophagy Induction

doi: 10.3389/fimmu.2020.607564

Figure Lengend Snippet: Synthesis of intermediate 1.

Article Snippet: Antibody against IL-1β (AB-401-NA) was purchased from R&D systems (Minneapolis, MN).

Techniques:

Journal: Frontiers in Immunology

Article Title: A Synthetic Small Molecule F240B Decreases NLRP3 Inflammasome Activation by Autophagy Induction

doi: 10.3389/fimmu.2020.607564

Figure Lengend Snippet: F240B inhibits the NLRP3 inflammasome-derived IL-1β secretion in J774A.1 macrophages. J774A.1 macrophages were incubated with 1 µg/ml LPS for 5 h followed by incubated with F240B for 0.5 h or 3 h. Cells then incubated with 5 mM ATP for 0.5 h (A, C) , 10 μM nigericin for 0.5 h (B, D) , 100 μg/ml MSU or 100 μg/ml SiO 2 for 24 h (E) . The levels of IL-1β and TNF-α in the supernatants were measured by ELISA. (F) J774A.1 macrophages were incubated with F240B for 3.5 h or 24 h, and cytotoxicity was analyzed by LDH release. The data are expressed as the mean ± SD of three separate experiments. * and *** indicate a significant difference at the level of p < 0.05 and p < 0.001, respectively, compared to LPS+ATP (A, C) , LPS+Nigericin (B, D) , LPS+MSU or LPS+SiO 2 (E) or control (F) .

Article Snippet: Antibody against IL-1β (AB-401-NA) was purchased from R&D systems (Minneapolis, MN).

Techniques: Derivative Assay, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Immunology

Article Title: A Synthetic Small Molecule F240B Decreases NLRP3 Inflammasome Activation by Autophagy Induction

doi: 10.3389/fimmu.2020.607564

Figure Lengend Snippet: F240B induces autophagy. (A, B) J774A.1 macrophages were incubated for 3–24 h with 0.3 µM F240B or for 4 h with 0.1 µM rapamycin. The levels of LC3 (A) and p62 (B) in the lysates were measured by Western blotting. (C) J774A.1 macrophages were incubated for 3 h with 0.1–1 µM F240B or for 4 h with 0.1 µM rapamycin. The levels of p62 in the lysates were measured by Western blotting. (D) GFP-LC3 expressed J774A.1 macrophages were incubated for 3 h with 1 µM F240B or for 4 h with 0.1 µM rapamycin. The LC3-GFP speck formation was measured by confocal microscopy. (E) J774A.1 macrophages were incubated for 3 h with 1 µM F240B. The cells were stained with 50 nM MDC or 1 µg/ml AO and the fluorescent signals were acquired by confocal microscopy.

Article Snippet: Antibody against IL-1β (AB-401-NA) was purchased from R&D systems (Minneapolis, MN).

Techniques: Incubation, Western Blot, Confocal Microscopy, Staining

Journal: Frontiers in Immunology

Article Title: A Synthetic Small Molecule F240B Decreases NLRP3 Inflammasome Activation by Autophagy Induction

doi: 10.3389/fimmu.2020.607564

Figure Lengend Snippet: Effect of F240B on inflammasome activation. (A) BMDM, (B) THP-1 macrophage or (C) PBMCs were incubated with 1 µg/ml LPS for 5 h followed by incubated with F240B for 3 h. Cells then incubated with 5 mM ATP or 10 μM nigericin for 0.5 h. The levels of IL-1β in the supernatants were measured by ELISA. (D) LPS-primed or Pam3CSK4-primed (for LPS transfection only) J774A.1 macrophages were incubated for 3 h with 1 μM F240B followed by transfection with poly(dA/dT) (2 μg/ml), FLA-ST (1 μg/ml), MDP (10 μg/ml) or LPS (2 μg/ml) for 6 h, or by Salmonella infection (20 MOI) for 2 h. The levels of IL-1β in the supernatants were measured by ELISA. The data are expressed as the mean ± SD of three separate experiments. *, ** and *** indicate a significant difference at the level of p < 0.05, p < 0.01 and p < 0.001, respectively, compared to LPS+ATP or LPS+Nigericin (A–C) or as indicated (D) .

Article Snippet: Antibody against IL-1β (AB-401-NA) was purchased from R&D systems (Minneapolis, MN).

Techniques: Activation Assay, Incubation, Enzyme-linked Immunosorbent Assay, Transfection, Infection

Journal: Frontiers in Immunology

Article Title: A Synthetic Small Molecule F240B Decreases NLRP3 Inflammasome Activation by Autophagy Induction

doi: 10.3389/fimmu.2020.607564

Figure Lengend Snippet: F240B inhibits the NLRP3 inflammasome through autophagy induction. (A, B) J774A.1 macrophages were incubated with 1 µg/ml LPS for 5 h followed by incubated with F240B for 3 h. Cells then incubated with 5 mM ATP for 0.5 h. The levels of IL-1β and caspase-1 (A) or NLRP3 and ASC (B) in the supernatants were measured by Western blotting. (C) J774A.1 macrophages were incubated with 1 µg/ml LPS for 5 h followed by incubated with 1 µM F240B in the presence or absence of 5 mM 3-MA for 3 h. Cells then were incubated with 5 mM ATP or 10 μM nigericin for 0.5 h. The levels of IL-1β in the supernatants were measured by ELISA. (D) Wild-type and LC3-knockout J774A.1 macrophages were incubated with 1 µg/ml LPS for 5 h followed by incubated with 1 µM F240B for 3 h. Cells then incubated with 5 mM ATP or 10 μM nigericin for 0.5 h. The levels of IL-1β in the supernatants were measured by ELISA. The data are expressed as the mean ± SD of three separate experiments. * and *** indicate a significant difference at the level of p < 0.05 and p < 0.001, respectively.

Article Snippet: Antibody against IL-1β (AB-401-NA) was purchased from R&D systems (Minneapolis, MN).

Techniques: Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Knock-Out

Journal: Frontiers in Immunology

Article Title: A Synthetic Small Molecule F240B Decreases NLRP3 Inflammasome Activation by Autophagy Induction

doi: 10.3389/fimmu.2020.607564

Figure Lengend Snippet: Activation of autophagy by F240B inhibits NF-κB activation and proIL-1β expression. (A) J774A.1 macrophages were incubated with F240B for 0.5 h followed by incubated with 1 µg/ml LPS for 6 h. The levels of proIL-1β and NLRP3 in the cell lysates were measured by Western blotting. (B) J-Blue cells were incubated with F240B for 0.5 h followed by incubated with 1 µg/ml LPS for 24 h. The NF-κB transcriptional activity was measured by NF-κB reporter assay. (C, D) J774A.1 macrophages were incubated with F240B (1 µM for ROS assay) for 0.5 h followed by incubated with 1 µg/ml LPS for 10 min. The phosphorylation levels of IκBα in the cell lysates were measured by Western blotting (C) , and the intracellular ROS production was analysed by H 2 DCFDA staining (D) . (E) J774A.1 macrophages were incubated with 1 µM F240B for 0.5 h followed by incubated with 1 µg/ml LPS for 10-30 min. The phosphorylation levels of ERK1/2, JNK1/2 and p38 in the cell lysates were measured by Western blotting. (F) Will-type or LC3-knockout J774A.1 macrophages were incubated with1 µM F240B for 0.5 h followed by incubated with 1 µg/ml LPS for 6 h. The levels of proIL-1β in the cell lysates were measured by Western blotting. ** and *** indicate a significant difference at the level of p < 0.01 and p < 0.001, respectively compared to LPS.

Article Snippet: Antibody against IL-1β (AB-401-NA) was purchased from R&D systems (Minneapolis, MN).

Techniques: Activation Assay, Expressing, Incubation, Western Blot, Activity Assay, Reporter Assay, ROS Assay, Phospho-proteomics, Staining, Knock-Out

Journal: Frontiers in Immunology

Article Title: A Synthetic Small Molecule F240B Decreases NLRP3 Inflammasome Activation by Autophagy Induction

doi: 10.3389/fimmu.2020.607564

Figure Lengend Snippet: Activation of autophagy by F240B reduced the protein stability of NLRP3 and proIL-1β. (A) J774A.1 macrophages were incubated with 1 µg/ml LPS for 5 h followed by incubated with 1 µM F240B or 0.1% DMSO for 3 h. The cells were then incubated with 30 µg/ml CHX for 3-12 h. The levels of NLRP3 and proIL-1β in the cell lysates were measured by Western blotting. (B) J774A.1 macrophages were incubated with 1 µg/ml LPS for 5 h followed by incubated with 1 µM F240B for 3 h in the presence or absence of 5 mM 3-MA. The cells were then incubated with 30 µg/ml CHX for 12 h. The levels of NLRP3 and proIL-1β in the cell lysates were measured by Western blotting.

Article Snippet: Antibody against IL-1β (AB-401-NA) was purchased from R&D systems (Minneapolis, MN).

Techniques: Activation Assay, Incubation, Western Blot

Journal: Frontiers in Immunology

Article Title: A Synthetic Small Molecule F240B Decreases NLRP3 Inflammasome Activation by Autophagy Induction

doi: 10.3389/fimmu.2020.607564

Figure Lengend Snippet: Activation of autophagy by F240B limits mitochondrial integrity loss. (A) J774A.1 macrophages were incubated with 1 µg/ml LPS for 5 h followed by incubated with 1 µM F240B in the presence or absence of 5 mM 3-MA for 3 h. Cells then incubated with 5 mM ATP for 0.5 h. (B) Wild-type or LC3-knockout J774A.1 macrophages were incubated with 1 µg/ml LPS for 5 h followed by incubated with 1 µM F240B for 3 h. Cells then incubated with 5 mM ATP for 0.5 h. The mitochondrial membrane integrity was measured by staining with MitoTracker Deep Red and MitoTracker Green. The percentage of cells with mitochondrial membrane integrity loss are expressed as the mean ± SD of three separate experiments. * and ** indicate a significant difference at the level of p < 0.05 and p < 0.01, respectively.

Article Snippet: Antibody against IL-1β (AB-401-NA) was purchased from R&D systems (Minneapolis, MN).

Techniques: Activation Assay, Incubation, Knock-Out, Membrane, Staining

Journal: Frontiers in Immunology

Article Title: A Synthetic Small Molecule F240B Decreases NLRP3 Inflammasome Activation by Autophagy Induction

doi: 10.3389/fimmu.2020.607564

Figure Lengend Snippet: F240B inhibits ASC oligomerization. (A) ASC-GFP expressed J774A.1 macrophages or (B) J774A.1 macrophages were incubated with 1 µg/ml LPS for 5 h followed by incubated with 1 µM F240B for 3 h. Cells then incubated with 5 mM ATP for 0.5 h. The ASC speck formation was analyzed by fluorescent microscope (A) , or the cell lysates were crosslinked by disuccinimidyl suberate and ASC oligomerization was analyzed by Western blotting (B) . (C–E) J774A.1 macrophages were incubated with 1 µg/ml LPS for 5 h followed by incubated with 1 µM F240B or 0.1 µM MCC950 for 3 h. Cells then incubated with 5 mM ATP for 0.5 h. The interaction between NLRP3 with ASC (C) , NEK7 (D) or PKR (E) were analyzed by immunoprecipitation and Western blotting assay. The percentage of ASC speck positive cells are expressed as the mean ± SD of three separate experiments. * indicates a significant difference at the level of p < 0.05.

Article Snippet: Antibody against IL-1β (AB-401-NA) was purchased from R&D systems (Minneapolis, MN).

Techniques: Incubation, Microscopy, Western Blot, Immunoprecipitation

Journal: Frontiers in Immunology

Article Title: A Synthetic Small Molecule F240B Decreases NLRP3 Inflammasome Activation by Autophagy Induction

doi: 10.3389/fimmu.2020.607564

Figure Lengend Snippet: F240B inhibits NO, COX-2 and IL-6 expression. RAW264.7 macrophages were incubated with F240B for 0.5 h followed by incubated with 1 µg/ml LPS for 24 h. The levels of COX-2 (A) in the cell lysates were measured by Western blotting, and the levels of NO (B) , IL-6 (C) and TNF-α (D) in the supernatants were measured by ELISA. The data are expressed as the mean ± SD of three separate experiments. ** and *** indicate a significant difference at the level of p < 0.01 and p < 0.001, respectively, compared to LPS.

Article Snippet: Antibody against IL-1β (AB-401-NA) was purchased from R&D systems (Minneapolis, MN).

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

Journal: Frontiers in Immunology

Article Title: A Synthetic Small Molecule F240B Decreases NLRP3 Inflammasome Activation by Autophagy Induction

doi: 10.3389/fimmu.2020.607564

Figure Lengend Snippet: F240B inhibits the NLRP3 inflammasome and inflammation in a mouse model of gouty inflammation. (A) Neutrophil and peritoneal lavage cells influx were quantified by Gr-1 and CD45 staining and cell count, respectively. (B, C) The expression levels of IL-1β, active caspase-1, IL-6 and MCP-1 in the peritoneal lavage fluids were measured by ELISA. The ELISA data expressed as mean ± SD of three separate experiments. *, ** and *** indicate a significant difference at the level of p < 0.05, p < 0.01 and p < 0.001, respectively, compared to MSU crystal-injected mice. ### indicates a significant difference at the level of p < 0.001 compared to control mice.

Article Snippet: Antibody against IL-1β (AB-401-NA) was purchased from R&D systems (Minneapolis, MN).

Techniques: Staining, Cell Counting, Expressing, Enzyme-linked Immunosorbent Assay, Injection, Control

Journal: Nature Communications

Article Title: MAP4K3 mediates amino acid-dependent regulation of autophagy via phosphorylation of TFEB

doi: 10.1038/s41467-018-03340-7

Figure Lengend Snippet: Knockout of MAP4K3 promotes autophagy induction and flux. a Validation of MAP4K3 knockout (k.o.) cell lines. Wild-type (WT) and HEK293A cells gene-edited with either of two different sgRNAs (M1 and M4) were lysed, and protein lysates were immunoblotted for MAP4K3. Immunoblotting of β-actin served as a loading control. b , c Knockout of MAP4K3 promotes autophagy flux. WT HEK293A cells, M1-1 MAP4K3 k.o. cells, and M4-6 MAP4K3 k.o. cells (not shown) were cultured in complete media (CM) or subjected to amino acid starvation (– AA), and remained untreated or were treated with ammonium chloride. Protein lysates were immunoblotted for LC3 and β-actin, which served as a loading control b . The ratio of LC3-II:actin was determined by densitometry using ImageJ and normalized to WT CM, which was arbitrarily set to 1 c . One-way ANOVA with post-hoc Tukey’s test; * P < 0.05. d , e Knockout of MAP4K3 promotes autophagy induction. LC-3 immunostaining of WT HEK293A cells and three different MAP4K3 k.o. cell lines, all cultured in CM d . Quantification of LC3 puncta area per cell area was determined using ImageJ. n > 100 cells per genotype. One-way ANOVA with post-hoc Tukey’s test; ** P < 0.01, *** P < 0.001. f – h Knockout of MAP4K3 promotes autophagy flux. WT HEK293A cells and MAP4K3 k.o. cells were cultured in CM or amino acid starved, and were transfected with a GFP-mCherry-LC3 expression construct ( f ). Note the predominance of red puncta indicative of autolysosomes in MAP4K3 k.o. cells. Quantification of autophagosome number per cell was determined by counting yellow puncta GFP-mCherry-LC3-expressing cell ( g ). Quantification of autolysosome number per cell was determined by counting red puncta/GFP-mCherry-LC3-expressing cell ( h ). n > 50 cells per condition. One-way ANOVA with post-hoc Tukey’ test; ** P < 0.01, *** P < 0.001. All experiments were performed in triplicate. Error bars = SEM. Scale bars = 10 μm

Article Snippet: Membranes were incubated overnight with primary antibodies against the following: LC3 (Novus Biologicals, NB100-2220) 1/3000; β-actin (Abcam, #ab8226) 1/10,000; Map4k3 (Cell Signaling, 9613) 1/1000; RagA (Cell Signaling, 4357) 1/1000; RagC (Cell Signaling, 5466) 1/1000; Lamtor1 (Cell Signaling, 8975) 1/1000; TFEB (Cell Signaling, 4240) 1/1000; pan 14-3-3 (Santa Cruz Biotechnology, sc-629) 1/1000; FLAG (M2) (Sigma; F1804); Raptor (24C12) (Cell Signaling, 2280) 1/1000; phospho-(Ser) 14-3-3 Binding Motif (Cell Signaling, 9601) (for detection of TFEB phosphorylation at Ser211) 1/1000; GST (Santa Cruz Biotechnology, sc-138) 1/1000, in 5% bovine serum albumin in PBS-T. Species-specific secondary antibodies were goat anti-rabbit IgG-HRP (Santa Cruz, sc-2004) or goat anti-mouse IgG-HRP (Santa Cruz, sc-2005), diluted 1/10,000 in 5% PBS-T milk and incubated for 1 h at RT.

Techniques: Knock-Out, Biomarker Discovery, Western Blot, Control, Cell Culture, Immunostaining, Transfection, Expressing, Construct

Journal: Nature Communications

Article Title: MAP4K3 mediates amino acid-dependent regulation of autophagy via phosphorylation of TFEB

doi: 10.1038/s41467-018-03340-7

Figure Lengend Snippet: Phosphorylation of TFEB at serine 3 is a key determinant of TFEB cellular regulation and autophagy function. a TFEB serine 3 phosphorylation is required for mTORC1 phosphorylation at serine 211. WT HEK293A and MAP4K3 k.o. cells were transfected with TFEB-FLAG, TFEB-S3A-FLAG, or TFEB-S3E-FLAG, and Torin1 treated. FLAG immunoprecipitates were immunoblotted and TFEB serine 211 phosphorylation as a fraction of total TFEB was quantified by densitometry. b MAP4K3 phosphorylation of TFEB is required for mTORC1 phosphorylation at serine 211. WT HEK293A and MAP4K3 k.o. cells were transfected with TFEB-FLAG and either no MAP4K3 (--), WT-MAP4K3, or KD-MAP4K3. FLAG immunoprecipitates were immunoblotted, and TFEB serine 211 phosphorylation as a fraction of total TFEB was quantified by densitometry. c TFEB serine 3 phosphorylation is required for interaction with 14-3-3. TFEB k.o. cells were transfected with no TFEB (--), inducible TFEB-WT-FLAG, or inducible TFEB-S3A-FLAG, and Torin1 treated, whereas all cells received doxycycline to induce TFEB-WT or TFEB-S3A expression. Cell lysates and TFEB immunoprecipitates were immunoblotted and immunoprecipitated 14-3-3 was quantified by densitometry. One-way ANOVA with post-hoc Tukey’s test; * P < 0.05, ** P < 0.01. d TFEB serine 3 phosphorylation regulates TFEB nuclear localization. TFEB k.o. cells were transfected with inducible TFEB-WT-FLAG or TFEB-S3A-FLAG, and cultured in CM or amino-acid starved (– AA). Under amino acid deprivation, TFEB localizes to nucleus, regardless of serine 3 status; however, upon amino acid satiety, mutation of TFEB serine 3 to phospho-resistant alanine prevents retention of TFEB in the cytosol. Quantification of TFEB nuclear localization to right. n > 100 cells per condition. One-way ANOVA with post-hoc Tukey’s test; *** P < 0.001. e TFEB serine 3 phosphorylation regulates autophagy activation. TFEB k.o. cells were transfected with GFP-mCherry-LC3 and either inducible TFEB-WT-FLAG or TFEB-S3A-FLAG, and cultured in CM and doxycycline, as indicated. Note the red puncta indicative of autolysosomes in cells expressing TFEB-S3A. Autophagosome number per cell was determined by counting yellow puncta per GFP-mCherry-LC3-expressing cell, and autolysosome number per cell was determined by counting red puncta per GFP-mCherry-LC3-expressing cell. n > 50 cells per condition. One-way ANOVA with post-hoc Tukey’s test; * P 0 < 0.05, ** P < 0.01. All experiments performed in triplicate. Error bars = SEM. Scale bars = 20 μm

Article Snippet: Membranes were incubated overnight with primary antibodies against the following: LC3 (Novus Biologicals, NB100-2220) 1/3000; β-actin (Abcam, #ab8226) 1/10,000; Map4k3 (Cell Signaling, 9613) 1/1000; RagA (Cell Signaling, 4357) 1/1000; RagC (Cell Signaling, 5466) 1/1000; Lamtor1 (Cell Signaling, 8975) 1/1000; TFEB (Cell Signaling, 4240) 1/1000; pan 14-3-3 (Santa Cruz Biotechnology, sc-629) 1/1000; FLAG (M2) (Sigma; F1804); Raptor (24C12) (Cell Signaling, 2280) 1/1000; phospho-(Ser) 14-3-3 Binding Motif (Cell Signaling, 9601) (for detection of TFEB phosphorylation at Ser211) 1/1000; GST (Santa Cruz Biotechnology, sc-138) 1/1000, in 5% bovine serum albumin in PBS-T. Species-specific secondary antibodies were goat anti-rabbit IgG-HRP (Santa Cruz, sc-2004) or goat anti-mouse IgG-HRP (Santa Cruz, sc-2005), diluted 1/10,000 in 5% PBS-T milk and incubated for 1 h at RT.

Techniques: Phospho-proteomics, Transfection, Expressing, Immunoprecipitation, Cell Culture, Mutagenesis, Activation Assay