Fk506

3F4 antibody (BioLegend, San Diego, USA) is a mouse monoclonal antibody recognizing the amino acid residues 109–112 of human PrP. Anti-ionized calcium-binding adapter molecule 1 (IBA1) antibody to detect microglia (Wako Pure Chemical Industries, Japan) and anti-glial fibrillary acidic protein (GFAP) antibody to detect astrocytes (DAKO, Japan) are rabbit polyclonal antibodies. FK506 (Selleck Chemicals, Houston, USA) was dissolved as previously described [22 (link)]. Briefly, FK506 was mixed with Kolliphor HS15 (gifted from BASF Japan), tetraglycol (Sigma-Aldrich, Japan), and ethyl oleate (Nacalai Tesque, Japan) and was stored at 25 °C. Doxycycline hyclate (Sigma-Aldrich) was dissolved in dH₂O and stored at − 20 °C.

MCF7 (HTB-22; ATCC) and HEK293T (632180; Takara) cells were cultured in Dulbecco's modified Eagle's medium (044-29765; WAKO) supplemented with 10% fetal bovine serum (FBS) (173012; Sigma) and an antibiotic-antimycotic solution (15240062; Thermo Fisher Scientific). HT29 (HTB-38; ATCC) and HCT116 (CCL-247; ATCC) cells were cultured in McCoy's 5A medium (16600-082; Gibco) containing 10% FBS and antibiotic-antimycotic solution. 22Rv1 (CRL-2505; ATCC) and LNCaP (CRL-1740; ATCC) cells were cultured in RPMI medium (187-02705; WAKO) containing 10% FBS and antibiotic-antimycotic solution. All cells were cultured at 37 °C under 5% CO₂. The cells were treated with FK506 (S500313; Selleckchem), MG132 (S261917; Selleckchem), and CN585 (207003; Merck). FK506 was used at concentrations of 50 μM, 25 μM, 12.5 μM or 6.25 μM, MG132 at 10 μM, and CN585 at 30 μM, 25 μM, 15 μM, or 7.5 μM. Cycloheximide (037-20991; FUJIFILM) was used at a concentration of 50 μg/ml. MLN4924 (S7109; Selleckchem) was used at a concentration of 0.5 μM. Okadaic acid (152-03271; FUJIFILM) was used at concentrations of 50 μM or 25 μM. AS1842856 (HY-100596; MedChemExpress) was used at a concentration of 40 μM, 20 μM, or 10 μM.

MCF7 (HTB-22, ATCC), T47D (HTB-133, ATCC), and HEK293T (632180, Takara) cells were cultured in Dulbecco's modified Eagle's medium (D-MEM) (044-29765, WAKO) supplemented with 10% fetal bovine serum (FBS) (173,012, Sigma) and antibiotic–antimycotic solution (15240062, Thermo Fisher Scientific). HT29 (HTB-38, ATCC) cells were cultured in McCoy's 5A medium (16600-082; Gibco) containing 10% FBS and antibiotic–antimycotic solution. All cells were cultured at 37 °C under 5% CO₂. Cells were treated with FK506 (S500313; Selleckchem), MG132 (S261917; Selleckchem), and CN585 (207003; Merck). FK506 was used at concentrations of 50, 25, or 12.5; MG132 at 10 μM; and CN585 at 30, 25, 15, or 7.5 μM. Cycloheximide (037-20991; FUJIFILM) was used at 50 μg/mL.

L-Kynurenine (L-Kyn) was purchased from Sigma (K8625), dissolved in DMSO, and used at 100μM. Cyclosporin A was purchased form Selleckchem (S2286), dissolved in DMSO, and used at 300nM. FK506 was purchased from Selleckchem (S5003) resuspended in DMSO, and used at 300nM. Anti-IgG (Sigma I5260) was used at 10μg/mL for 30 minutes to overnight. Control conditions were treated with equal amounts of DMSO.

Src-kinase inhibitor PP2 (Selleck Chemicals) and Lck-inhibitor II (EMD Millipore) were used. Inhibitors were suspended in DMSO and cells treated with DMSO alone served as controls. Unless otherwise noted, to achieve optimum inhibition of kinase activity, Jurkat T cells and primary PBMCs were treated with PP2 at concentrations of 5μg/ml and 1μg/ml respectively. Lck-inhibitor II was used at 10μg/ml. Calcineurin inhibitors FK506 and Cyclosporin A (CsA) were obtained from Selleck Chemicals and were used at 1μM concentration. Cells were treated with the inhibitors for 18 hours before stimulation.

Animals were treated with FK506 at a dose of 3 mg/kg/day (Muthuraman and Sood, 2010 (link)), similar to a previous study (Yoshiyama et al., 2007 (link)). Specifically, FK506 (Selleck Chemicals) was solubilized in DMSO at a concentration of 9 mg/mL to make stock solution aliquots. The FK506 stock was solubilized into sucrose-supplemented (2%) drinking water in mice at a dilution of 1:400 (bringing the final DMSO concentration to 0.25%) and changed every other day. Considering animals drink ~4 mL/day, they are expected to consume 0.09 mg/day via this passive administration route, or the equivalent of 3 mg/kg at an average weight of 30 g. Analysis of AFSM and gliosis in FK506-treated animals was performed as mentioned above.

Female NOD/Shi-SCID mice (CLEA Japan Inc.) were maintained under specific pathogen-free conditions and fed a sterilized standard diet (CE-2, CLEA Japan Inc.). HT29 cells (2 × 10⁶ cells/100 μL PBS per site) were subcutaneously transplanted into the left flank. FK506 (S500313, Selleck Chem) treatment was initiated when the tumor size reached 150 mm³. Mice intraperitoneally received 3 mg/kg/day FK506 or vehicle control (corn oil) once daily for 3 weeks. From dissected mice, tumor fragments were harvested and homogenized in radioimmunoprecipitation assay buffer (50 mM Tris–HCl at pH 7.5, 150 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 10% sodium deoxycholate, 10% SDS, and 1% Triton X-100) supplemented with phosphatase inhibitors and protease inhibitors. The supernatant extracted by sonication was mixed with sample buffer and boiled for 5 min. The animal experiments were approved by the Committee for Animal Use of Yamaguchi University. All methods were carried out in accordance with relevant guidelines and regulations. All studies are performed in accordance with ARRIVE guidelines.