Fig 2: 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

Fig 3: Exosomal TRIM59 is characteristically secreted by lung cancer cells and transferred to and internalized by macrophages via exosomes. a. Exosomes released by H1299 and A549 cells were detected by electron microscopy. Scale bar, 100 nm. Immunoblotting assay of indicated proteins in exosomes from H1299 and A549. b. Western blot evaluations were used to evaluateTRIM59 expression in both LC cells (H1299 and A549) exosomes and LC cells treated with TRIM59 shRNA or scrambled controls. c-d. Representative immunofluorescence image shows the internalization of PKH67-labeled H1299-derived exosomes (green) by macrophages. Confocal imaging showed the delivery of PKH67-labeled exosomes (green) to macrophages. Green dots represented delivered exosomes. Scale bar, 150 μm. e-f. THP-1 macrophages cells were incubated with exosomes for the noted periods of time or the noted doses. Western blot evaluations were used to evaluate TRIM59

Fig 4: TRIM59 stimulates macrophages to facilitate tumor growth and metastasis in vivo. a. Representative image of tumor growth in WT and TgTRIM59 mice subcutaneously inoculated with LLC cells (n = 5 per group). b. The growth curve of subcutaneous tumor in WT and TgTRIM59 mice. And comparison of tumor weight from two groups at the end of the experiment. c. LLC cells (5.0 × 106/100 μl of DMEM) were intravenously injected into WT and TgTRIM59 mice via the tail vein (n = 6 in each group). Mice were sacrificed for further observation on day 14. Representative images of lung metastasis in WT and TgTRIM59 mice. The total area of invasive lesions on the lung slice section represents the invasive tumor volume in the lungs. Immunoblotting assay of TRIM59 in TAMs from the lung metastases of WT or TgTRIM59 mice. Scale bars, 200 μm. d. Fluorescent multiplex immunohistochemistry (mIHC) staining of adjacent noncancerous tissues and LC tissues. Representative image of an LC case was shown with TRIM59 and IL-1β co-expression (DAPI, blue; TRIM59, red; IL-1β, green; CD68, yellow). Scale bars, 200 μm. e. multiplex immunohistochemistry analysis of TRIM59 and IL-1β protein levels in lung cancer samples on tissue microarrays. The expression of TRIM59 and IL-1β expression in TAMs were measured with mean fluorescence intensities (MFIs) (in arbitrary units, a.u.), respectively. The Pearson correlation between TRIM59 and IL-1β expression (n = 90; p < 0.01, r = 0.414). f. Chematic illustration of the crosstalk between macrophages and cancer cells in the tumor microenvironment. Our data indicate that exosomal TRIM59 stimulates macrophages NLRP3 inflammasome activation to release IL1-β, which, in turn, promotes LC cells proliferation and invasion. All data are shown as mean ± SEM. Student’s t-test was used to analyze the data. **p < 0.01

Fig 5: 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