Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cancer-derived exosomal TRIM59 regulates macrophage NLRP3 inflammasome activation to promote lung cancer progression

doi: 10.1186/s13046-020-01688-7

Figure Lengend Snippet: TRIM59 binds and co-localizes with ABHD5. a . Network graph representation of interaction from the BioGRID for TRIM59. Users can select the ‘Network’ tab from the ‘Switch View’ menu to view interactions data when available. b . HEK-293 cells were transfected with HA-tagged TRIM59 and Myc-tagged ABHD5, lysates were prepared and immunoprecipitated (IP) with either anti-HA or anti-Myc, and the precipitates were evaluated using immunoblot (IB) analysis as noted. c - e . Immunohistochemical demonstration of the co-localization of TRIM59 and ABHD5 in THP-1 macrophages, BMDMs, and TAMs. Fluorescence images were counterstained with 4′6-diamidino-2-phenylindole (DAPI) for nucleus identification. Scale bars, 10 μm. f . THP-1 macrophages cells were transiently transfected with HA-tagged TRIM59 and Myc-tagged ABHD5. Rabbit anti-HA and Mouse anti-Myc antibody were used for the proximity ligation assay. Red dots present the interaction of TRIM59 with ABHD5. Scale bars, 20 μm

Article Snippet: HA-TRIM59 (Cat: HG25849-NY), TRIM59 (Cat:HG25849-UT), Myc-ABHD5 (Cat: HG14216-CM), and ABHD5 (Cat: HG14216-UT) expression plasmid were purchased from Sino Biological Inc.

Techniques: Transfection, Immunoprecipitation, Western Blot, Immunohistochemical staining, Fluorescence, Proximity Ligation Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cancer-derived exosomal TRIM59 regulates macrophage NLRP3 inflammasome activation to promote lung cancer progression

doi: 10.1186/s13046-020-01688-7

Figure Lengend Snippet: TRIM59 promotes ABHD5 protein degradation through ubiquitination. a . Proteasome inhibitor MG132 inhibited the downregulation of ABHD5 protein levels induced by HA-TRIM59 expression in HEK-293 cells. HEK-293 cells were transfected with HA-TRIM59 or HA-TRIM59-∆RING vector. The indicated proteins were measured by western blotting. b . HA-TRIM59 expression decreased ABHD5 protein half-life in cells. HEK-293 cells with ectopic HA-TRIM59 expression and control cells were transfected with the Myc-ABHD5 vector. The cells were treated with 50 μg/ml CHX for indicated time periods before being collected for western blot assays. c . Knockdown of endogenous TRIM59 increased Myc-ABHD5 protein half-life in THP-1 macrophages. d . The effects of expression of HA-TRIM59 and its mutants on ubiquitination of Myc-ABHD5 in HEK-293 cells analyzed by in vivo ubiquitination assays. e . Knockdown of endogenous TRIM59 decreased the ubiquitination of Myc-ABHD5 in THP-1 macrophages analyzed by in vivo ubiquitination assays. f . Mutations of TRIM59 that impaired TRIM59’s ubiquitination activity impaired the ability of HA-TRIM59 to degrade Myc-ABHD5 protein in THP-1 macrophages

Article Snippet: HA-TRIM59 (Cat: HG25849-NY), TRIM59 (Cat:HG25849-UT), Myc-ABHD5 (Cat: HG14216-CM), and ABHD5 (Cat: HG14216-UT) expression plasmid were purchased from Sino Biological Inc.

Techniques: Expressing, Transfection, Plasmid Preparation, Western Blot, In Vivo, Activity Assay

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cancer-derived exosomal TRIM59 regulates macrophage NLRP3 inflammasome activation to promote lung cancer progression

doi: 10.1186/s13046-020-01688-7

Figure Lengend Snippet: ABHD5 deficiency promotes NLRP3 inflammasome activation in macrophages. a . Western blot analysis of ABHD5 expression in macrophages transfected with scrambled control shRNA or ABHD5 shRNA for 36 h. Immunoblot of the IL-1β, the pro-caspase-1 and cleaved caspase-1 in the supernatants (SNs) or cell lysates of ABHD5-silenced THP-1 macrophages, primed with LPS, and then stimulated with Nigericin (Nig.), ATP or Alum. β-actin served as a loading control. Quantification of Western blotting were performed with the Image J software. Numbers below each blot indicate relative band intensity normalized to β-actin. b . ELISA of IL-1β in supernatants from THP-1 macrophages silenced of ABHD5, primed with LPS for 8 h, and followed by stimulation with ATP, Nig., Alum, poly(dA:dT) or flagellin for 30 min. c . ELISA of IL-1β in supernatants from BMDMs silenced of ABHD5, primed with LPS for 8 h, and followed by stimulation with ATP, Nig., Alum, poly(dA:dT) or flagellin for 30 min. d . RT-PCR analysis of IL-1β mRNA expression in macrophages transfected with shRNA as indicated and stimulated as indicated. e. ELISA of IL-1β in supernatants from THP-1 cells infected with lentiviral vectors expressing ABHD5 or GFP control, primed with LPS for various times, and followed by stimulation with ATP for 30 min. f . ELISA of IL-1β in supernatants from THP-1 cells infected with lentiviral vectors expressing TRIM59 or GFP control, primed with LPS, and followed by stimulation with ATP or Nig. Data are from three independent experiments with biological duplicates in each (mean ± SEM) or are representative of three independent experiments. Student’s t -test, ** p < 0.01

Article Snippet: HA-TRIM59 (Cat: HG25849-NY), TRIM59 (Cat:HG25849-UT), Myc-ABHD5 (Cat: HG14216-CM), and ABHD5 (Cat: HG14216-UT) expression plasmid were purchased from Sino Biological Inc.

Techniques: Activation Assay, Western Blot, Expressing, Transfection, shRNA, Software, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Infection

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cancer-derived exosomal TRIM59 regulates macrophage NLRP3 inflammasome activation to promote lung cancer progression

doi: 10.1186/s13046-020-01688-7

Figure Lengend Snippet: Loss of ABHD5 causes metabolic reprogramming in macrophages. a . THP-1 cells treated with shCtrl or shABHD5 and control were stained by oil red, representative photographs were taken, then analyzed lipid content by a quantitative method. Scale bars, 50 μm. b . THP-1 macrophages infected with lentiviral vectors expressing TRIM59 or GFP control and control stained by oil red, then analyzed lipid content by a quantitative method. Scale bars, 50 μm. c - d . THP-1 cells stably transfected with shCtrl or shABHD5 ( c ) or infected with TRIM59 lentiviral particles or GFP lentiviral control ( d ). Lactate secretion ability was measured. e - f . Protein level of MCT4 was analyzed by immunoblot. All statistic data in this figure represent mean ± SEM. The significance was determined by Student’s t -test. ** p < 0.01

Article Snippet: HA-TRIM59 (Cat: HG25849-NY), TRIM59 (Cat:HG25849-UT), Myc-ABHD5 (Cat: HG14216-CM), and ABHD5 (Cat: HG14216-UT) expression plasmid were purchased from Sino Biological Inc.

Techniques: Staining, Infection, Expressing, Stable Transfection, Transfection, Western Blot