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  • br Nuclear protein extraction and

    2020-08-30


    Nuclear protein extraction and western blot Intestinal epithelial cells (IEC) were re-suspended in buffer A (10mM HEPES (pH = 7.9), 10mM KCl, 10mM MgCl2, 1mM DTT and 1x protease inhibitor cocktail (Roche)) and incubated on ice for 15 min followed by adding 10% NP40 to total concentration of 1%. Content was vortexed for 20 s and spun down at 12000 rpm for 30 s. Supernatants were collected as cytoplasmic protein extracts. Pellets were re-suspended again in buffer A and centrifuged. Washed pellets were re-suspended in buffer C (20mM HEPES (pH = 7.9), 25% Glycerol, 0.4M NaCl, 1.5mM MgCl2 and 1x protease inhibitor cocktail) and incubated on ice for 15 min, then centri-fuged at 12000 rpm for 5 min. Supernatants were collected as nuclear protein extract, mixed with RIPA buffer with protease inhibitors and subjected to western blot analysis with anti-NF-kB p65 (Cell Signaling) antibody and anti-Histone H3 (Cell Signaling) antibody for loading control.
    In Vivo Treatment with Anakinra
    Mice were injected intraperitoneally for three consecutive days with 2.5 mg recombinant human IL-1Ra (Anakinra/Kineret, Rx, FCCC Pharmacy), or PBS for control group.
    RNA extraction and quantitative Real Time-PCR Analysis
    Total RNA was extracted with the RNAeasy Plus kit (QIAGEN) and reversely transcribed using IScript kit (Biorad). Quantitative RT-PCR (qRT-PCR) was performed with iTaq Universal SYBR Green Supermix (Biorad) on a Biorad CFX96 machine. Expression data were normalized to RpL32 mRNA levels. The data are presented in arbitrary units and were calculated as 2(Ct (RpL32 – gene of interest)). Primer sequences generally were obtained from the NIH Mouse qPrimerDepot website repository and are listed in Key Resources Table.
    Analysis of Cytokine Production by Multiplex or ELISA
    Small pieces of tumors were incubated for 24h in complete RPMI 1640 media. Supernatants were collected and cytokine secretion was measured by mouse Procarta 17-Plex cytokine array (eBioscience) on MagPix instrument (Luminex) according to manufac-turer’s instructions. For ELISA analysis of IL-17A, colonic tumors were cultured in DMEM containing 10% fetal bovine serum (FBS) and 1 , 2Dioleoyl-sn-glycero3PC overnight, and supernatant was analyzed with IL-17A ELISA kit (eBioscience). Cytokine concentration was normalized to the weight of tumors in each well.
    Immunohistology and immunofluorescence
    For immunohistology, immediately after euthanasia, colon rolls were fixed with 10% buffered formalin and kept overnight at RT. After fixation samples were transferred in 70% ethanol prior to paraffin embedding. 5 mm thick sections were cut in the sagital plane for staining with hematoxylin and eosin. Images were acquired and quantified with slide scanning Vectra microscope (Vectra).
    Sections were de-paraffinased in 4 changes of xylene, then dehydrated and re-hydrated in 100%->70% ethanol changes. Antigen retrieval of samples was performed in 1x Citrate buffer (10mM Citrate, 0.05% Tween 20, pH 6.0) at 95C for 1h. Then slides were rinsed in dH2O, dehydrated in 100% ethanol, then blocked with 3% H2O2 in PBS, rinsed with water, blocked in PBS-5% BSA-5% normal goat serum for 20 min, then incubated with primary antibody anti-Ki-67 (1:250) or anti-p-STAT3 (1:100) overnight at 4C, then washed 3x in PBS - 1% BSA. Next, secondary biotinylated anti-rabbit antibodies were applied at 1:200 dilution and incubated at RT for 1h, rinsed in PBS. Next, Streptavidin-HRP was applied at dilution 1:250 in PBS-1%BSA for 30 min at room temperature, slides were washed 3x in PBS, then developed with chromogen DAB solution for 7 min at room temperature. Next, slides were rinsed and counterstained with hematoxylin, rinsed with water and immersed into 0.25% ammonium hydroxide, dehydrated with 4 changes of ethanol 70%->100%, followed by 4 changes of xylene for 5 min each, and mounted.
    Images were acquired with slide scanning Vectra microscope (Vectra). Immunofluorescencent staining was performed in the same way with IHC protocol with few exceptions- blocking buffer was containing 5% goat serum, 10% BSA, 0.1% Triton X-100 in 1xPBS. Goat-anti-rabbit AF488 was used for secondary antibody, and then samples were washed follow by staining with DAPI for 10 min at RT. Slides were mounted with VectorShield and covered with coverslip. After drying for 1-2h at RT slides were analyzed on Leica SP8 Confocal Microscope.
    Whole tissue IF staining for bacteria with Yo-Yo 1 dye
    For whole-mount staining, colons were fixed in buffered 4% paraformaldehyde for 8h at 4C. Tissues were permeabilized by incuba-tion with 0.5% (wt/vol) saponin, 2% (vol/vol) FBS, and 0.09% (wt/vol) sodium azide in PBS for at least 18 hours. The same buffer was used for subsequent incubations with antibodies. Colon fragments were incubated with AlexaFluor 647 phalloidin (1:200) and Yo-Yo1 (1:500) dye for 12-16 hours at 4C followed by 1-2 hour incubation at 37C. Samples were washed with PBS, mounted flat in PBS and imaged with water dipping lens by Leica SP8 Confocal microscope.