Fetal calf serum (fcs)

C57BL/6NCrSlc (C57BL/6N) mice were purchased from SLC. Mice lacking IFN-γR have been previously described (30 (link)). All animal experiments were conducted with the approval of the Animal Research Committee of Research Institute for Microbial Diseases at Osaka University. RHΔhxgprtΔku80 and its derivatives of T. gondii were maintained in Vero cells by bi-weekly passage in RPMI (Nacalai Tesque) supplemented with 2% heat-inactivated fetal calf serum (FCS; JRH Bioscience), 100 U/mL penicillin and 0.1 mg/mL streptomycin (Nacalai Tesque). Mouse embryonic fibroblasts were maintained in Dulbecco’s modified Eagle medium (DMEM; Nacalai Tesque) supplemented with 10% heat-inactivated FCS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin (Nacalai Tesque).

Two PROS fibroblast cell lines were established from the patient's lower limb and perineum. Two control fibroblast cell lines (C2 and C3), which were previously established from the skins of two unrelated healthy volunteers of a 36-year-old male and a 27-year-old female, respectively, [62 (link)] were used. Additionally, a normal human dermal fibroblast cell line established from the skin of a 14-year-old male (NHDF-c, c-12300) was purchased from PromoCell GmbH (Heidelberg, Germany). Fibroblast cells were cultured as previously described [62 (link)]. Briefly, fibroblasts were maintained in Eagle's minimal essential medium (EMEM; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% FCS, penicillin (100 U/mL), streptomycin (0.1 mg/mL), L-glutamine (1.75 mM), HEPES (10 mM), and NaHCO₃ (0.13%). All supplemental reagents, except for FCS, were purchased from Nacalai Tesque (Kyoto, Japan). The cells were plated on 35-mm collagen-coated dishes at a density of 1.1 × 10⁴ cells/cm² for biochemical experiments. Fibroblast cells were treated with rapamycin (LKT Laboratories, St. Paul, MN, USA), NVP-BEZ235 (Chemscene, Monmouth Junction, NJ, USA), aspirin (acetylsalicylic acid; Wako, Osaka, Japan) and metformin hydrochloride (Sigma-Aldrich) for 2 days, washed with PBS, and then harvested and subjected to SDS-PAGE and western blotting.