Maraviroc

To determine the effects of the inhibitors on tumor cell viability, they were added to cancer cells for the same time periods used in ELISA assays and transwell invasion experiments (described above). Then, the cells were counted by trypan blue exclusion assay in ≥2 replicates/treatment, as described above. The selected concentrations of all inhibitors did not affect tumor cell viability. The only exception was the CCR2 inhibitor (see below), causing ~40% cell death in the lowest concentration possible. Therefore, in all ELISA assays performed with inhibitors, data were presented after normalization of protein amounts to cell numbers.
The following inhibitors were used in the study: (1) i-Gαi = Gαi inhibitor—Pertussis Toxin (PTx; 0.1–0.2 µg/mL; #516560, Merck); (2) i-Ras = Ras inhibitor—farnesylthiosalicyclic acid (FTS, Salirasib; 7 µM; #SML1166, Sigma-Aldrich); (3) i-CXCR1/2 = CXCR1/2 inhibitor—Reparixin (30–40 µM; #A12383, ADOOQ Bioscience, Irvine, CA, USA); (4) i-CCR2 = CCR2 inhibitor—CAS 445479-97-0 (0.02 µM; #227016, Merck); (5) i-CCR5 = CCR5 inhibitor—Maraviroc (30 µM; #14641, Cayman Chemical, Ann Arbor, MI, USA). All inhibitors were dissolved in DMSO; thus, this vehicle was used as control in all assays using the inhibitors.

The human prostatic carcinoma cell lines, PC-3 and LNCaP, were purchased from American Type Culture Collection and cultured in RPMI-1640 medium (Gibco, USA) containing 10% fetal bovine serum (FBS, HyClone, USA), 100 Units/ml penicillin and 0.1 mg/ml streptomycin. Human primary cultures of bone marrow-derived MSCs (BM-MSCs) were purchased from Stemcell Technologies (Vancouver, BC) and cultured using the Human MesenCult® Proliferation Kit (Stemcell Technologies Inc). The adherent cells were cultured in a 5% CO₂ humidified environment at 37 °C and the medium was changed every 2–3 days. Only cells from passages three to six were used for the experiments.
An antagonist of CCR5 (Maraviroc) was purchased from Cayman Chemicals (Ann Arbor, Michigan, USA), and another antagonist of CCR5 (TAK-779) was synthesized by Takeda Chemical Industries (Osaka). Notch1 inhibitors LY3039478 and IMR-1 were obtained from Selleck (Houston, TX, USA).

Eosinophils were isolated with >98% purity from human peripheral blood samples of healthy volunteers with mild eosinophilia (approximately 4–8% of total white blood cells) using negative selection via a MACS system and an eosinophil isolation kit (Miltenyi Biotec, Bergish Gladbach, Germany). After erythrocyte lysis, leukocytes were collected from the human peripheral blood sample and used in some experiments. Furthermore, the human bronchial epithelial cell line BEAS-2B was obtained from the European Collection of Authenticated Cell Culture (Salisbury, UK) and coincubated with purified eosinophils overnight (for 20–24 h), as appropriate. Subsequently, the eosinophils were stimulated with recombinant human CCL4 (Abcam, Cambridge, UK) and pretreated with CCR5 antagonist (Maraviroc; Cayman chemical, Ann Arbor, MI, USA), PDGFRβ inhibitor (Imatinib; Selleck, Houston, TX, USA), or Src family inhibitor (Dasatinib; Selleck), as appropriate. This study was approved by the local ethics committee of Kansai Medical University (approval number: KanIRin1313). All participants in this study provided written informed consent.

WT mice were i.p. injected with 20 μg of Maraviroc (14641; Cayman). 4 h later, the mice were immunized i.n. with 40 μl of 3 μg OVA-AF488 and 20 μg of CpG. After 24 h, LLNs were harvested and analyzed for Ag⁺-positive myeloid cells.

A total of 10⁴ monocytes or neutrophils were FACS-sorted (as above; purity, >98%) from mice primary immunized and challenged with Lm-Ova 6 weeks later for 16 hours. Cells were next coincubated in 96-well round-bottom plate and complete medium, with HKLm, rCCL3, rCCL4, rXCL1, or the combination of the three recombinant chemokines, in the presence of GolgiPlug/Stop for 4 hours at 37°C before staining for cell surface and intracellular markers for FACS analysis. In CCR5 and CCR1 blocking experiments, cells were incubated with both CCR5 (1 μM; Maraviroc, Cayman Chemical Company) and CCR1 (1 μM; J113863, Santa Cruz Biotechnology) chemical inhibitors or control dimethyl sulfoxide 30 min before adding recombinant chemokines or HKLm.

Molecular biology grade DMSO was purchased from Sigma Aldrich (St. Louis, MO, USA), Sterile PBS was from ThermoFisher Scientific (Waltham, MA, USA). 3CLpro inhibitor screening enzymatic assay kits (Catalog #79955-1) were from BPS Biosciences (San Diego, CA, USA). Amprenavir, Atazanavir sulfate, Candicin, Chloroquine Phosphate, Hydroychloroquine Sulfate, and Lopinavir were purchased from Sigma Aldrich (Saint Louis, MO). Beclabuvir, Temsavir were from Medchem Express (Monmouth Junction, NJ). Abacavir (sulfate), Arbidol hydrochloride, Asunaprevir, Atrovastatin, Boceprevir, Daclatasvir, Danoprevir, Darunavir, Delavirdine (mesylate), Edoxudine, Elbasvir, Elvitegravir, Etravirine, Favipiravir, Fumagillin, Glecaprevir, Grazoprevir, Indinavir sulfate, Itraconazole, Ivermectin, Ledipasvir (G-5885), Maraviroc, Methylprednisolone, micafungin sodium, ombitasvir, Oseltamivir phosphate, Paritaprevir, Peramivir, Pibrentasvir, Pimodivir, Pleconaril, Posaconazole, Quinine, Raltegravir (potassium salt), Remdesivir, Ribavirin 5’-monophosphate (lithium salt), Ribostamycin sulfate, Rilpivirine, Saquinavir, Telaprevir, Tenofovir diphosphate (sodium salt), and Velpatasvir were purchased from Cayman Chemicals (Ann Arbor, MI).