Nystatin

Analytical grade (to enable its use without further purification) rhodamine 123 (R123); sodium dodecyl sulfate (SDS); 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide (MTT); Dulbecco’s Modified Eagle’s medium–high glucose (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin mixture; resazurin sodium salt; trypsin-EDTA solution; allantoin; and tariquidar and dimethyl sulfoxide (DMSO) were acquired at Sigma-Aldrich (St. Louis, MO, USA). Doxorubicin hydrochloride was acquired from Teva Pharmaceuticals (Petah Tikva, Israel). Eagle’s Minimal Essential Medium (EMEM, Sigma-Aldrich) containing 4500 mg/L glucose, supplemented with a non-essential amino acid (NEAA) mixture (Sigma-Aldrich); a selection of vitamins and 10% heat-inactivated FBS; 2 mM L-glutamine (Sigma-Aldrich); 1 mM Na-pyruvate (Sigma-Aldrich); nystatin (Sigma-Aldrich); a penicillin-streptomycin mixture at concentrations of 100 U/L and 10 mg/L; RPMI 1640 medium (Sigma-Aldrich), supplemented with 10% FBS; 2 mM L-glutamine; 1 mM Na-pyruvate; 100 mM HEPES (Sigma-Aldrich); nystatin; and a penicillin-streptomycin mixture at concentrations of 100 U/L and 10 mg/L were used in the biological evaluation. Pgp-Glo™ Assay Systems (Promega), and an Annexin V-FITC Apoptosis Detection Kit were used (Calbiochem, EMD Biosciences. Inc. La Jolla, CA, USA).

Nystatin (Sigma N4014) was resuspended in DMSO to a concentration of 10 mg/mL. Mice were injected with 50 μL of 10 mg/mL Nystatin per footpad 1 hr prior to injection with ova conjugated to Alexa 488 (5 μg) in a mixture with polyI:C and anti-CD40 (2.5 μg each). LNs were harvested and digested as below (preparation of single-cell suspensions) and stained with CD45 brilliant violet 510 (Biolegend clone 30F11, 1:300), PDPN APC (Biolegend clone 8.1.1, 1:200), CD31 PercP Cy5.5 (Biolegend clone 390, 1:200), and PD-L1 pacific blue (Biolegend clone 10F.9G2, 1:200).

Isolation of C. abortus was performed in HEp-2 (Human Epithelial type 2) cells as follows. A pooled sample of placental cotyledons was ground up in 4 ml sucrose–phosphate–glutamate transport medium [21 ] using a mortar and pestle and sterile sand then centrifuged at 100 x g. The supernatant was collected before consecutive passages through 0.8 and 0.45 μm filters (Sterile Acrodisc^® Syringe Filters, Ann Arbor, USA). After filtration, the sample was inoculated onto HEp-2 cells grown on coverslips in Trac bottles (Thermo Scientific, Newport, UK) in Iscove’s Modified Dulbecco’s Medium (IMDM, Thermo Fisher Scientific, New York, USA), supplemented with 2% heat-inactivated foetal calf serum (Merck Life Science UK Limited, Gillingham, UK), 50 μg/mL gentamicin, 200 μg/mL streptomycin, 25U/mL nystatin and 1 μg/mL cycloheximide (Merck Ltd., Poole, UK). Trac bottles were centrifuged at 3,000 × g at room temperature for 2 × 15 min and incubated at 37°C in 5% CO₂ for 2 h before replenishing with fresh medium and then maintained under the same conditions. After 72 h, coverslips were fixed in methanol, stained using Giemsa ‘Gurr’ (Merck Ltd., Poole, UK), and examined for the presence of chlamydial inclusions by light microscopy.

Rabbit antibody to CCHFV N was generously provided by Ali Mirazimi (Karolinska Institutet, Sweden), and mouse antibody to CCHFV N was provided by Connie Schmaljohn (U.S. Army Medical Research Institute of Infectious Diseases, Washington, D.C.). The mouse antibody to CD63, clone H5C6, developed by J. Thomas August and James E.K. Hildreth (Johns Hopkins University School of Medicine) was obtained from the Developmental Studies Hybridoma Bank, created by the National Institute of Child Health and Human Development (NICHD), and maintained at The University of Iowa, Department of Biology, Iowa City. Mouse antibodies to Tsg101 and rabbit antibodies to Vps24, Vps4B, and PDCD6 (referred to as Alix/Aip1) were from Abcam, Cambridge, MA. Other antibodies used here were mouse antibody to GAPDH (Life Technologies, Carlsbad, CA), mouse antibody to EEA1 (BD Biosciences, Franklin Lakes, NJ), mouse antibody to Lamp1 (Santa Cruz Biotechnologies, Dallas, TX), and rabbit antibody to CCHFV Gc (IBT Bioservices, Gaithersburg, MD).
Dimethylsufoxide (DMSO) was from ATCC (Manassas, VA); 5-(N-Ethyl-N-isopropyl) amiloride (EIPA) was from Sigma (St. Louis, MO); LY294002, U18666A, bafilomycin A, dynasore, nystatin, and chlorpromazine hydrochloride (CPZ) were from EMD Millipore (Billerica, MA).

Tumor and adjacent normal tissue samples were obtained from patients with colorectal adenocarcinoma recruited at the Western Health Hospital Footscray, Eastern Health Hospital Box Hill, Northern Health Hospital Epping, and Royal Melbourne Hospital Parkville in Australia between 2017 and 2020. This study was conducted in accordance with the Declaration of Helsinki, the NHMRC Statement on Ethical Conduct in Human Research, and Institutional Human Research Ethics approval (HREC 2016.249, Walter and Eliza Hall Institute of Medical Research). All patients gave informed consent. Tumoroids and organoids were generated from respective tumor tissue and adjacent normal tissue from the resection margin from patients undergoing resection of primary tumors. For patients undergoing resection of metastatic disease, tumoroids were generated from tissue taken from the site of the metastasis. Specimens with a volume of greater than 5 mm³ were collected at surgery and placed into a collection medium containing DMEM/F12 (Life Technologies, 11320082), 250 U/ml penicillin-streptomycin (Life Technologies, 15140122), 50 µg/ml gentamycin (Merck, G1397), 100 µg/ml kanamycin (Merck, K0254), 2.5 µg/ml amphotericin B (Merck, A2942) and 100 U/ml nystatin (Merck, N1638), and stored for up to 72 h at 4 °C before processing.

For inflammasome activation, MDMs or THP1 cells were primed with 1 µg/mL LPS (lipopolysaccharide from Escherichia coli O26:B6, Sigma) in complete media for 3 hrs, washed with serum-free RPMI media, and treated with 10 µM nigericin for 45 mins or the Membrane Attack Complex (MAC) for the indicated time in serum free RPMI media. To form the MAC, cells were treated for 15 mins with 10 µg/mL C5b6, unless otherwise specified, and 10 µg/mL anti-CD59 mAb followed by treatment with 10 µg/mL C7, 10 µg/mL C8 and 10 µg/mL C9 (Complement Technologies). NLRP3 inflammasome activation was impaired using 1 µM MCC950 (Pepreotech). GSDMD processing was blocked using 10 µM NSA (Calbiochem). Endocytosis was blocked with 0.1 µg/mL nystatin (Merck), 10 µM cytochalasin D (Merck), or 10 µM dynasore (Calbiochem), unless otherwise specified. All inhibitors were used for 30 mins before and during stimulation with nigericin or the MAC in serum free RPMI media.