Concanavalin a (cona)

PBL from patients and controls were isolated via centrifugation on a Ficoll-Hypaque gradient for 20 min, washed twice, and resuspended in Roswell Park Memorial Institute 1640 medium with HEPES, supplemented with 2 mM glutamine (Sigma-Aldrich Japan, Tokyo, Japan), 100 units/ml penicillin G (Sigma-Aldrich Japan, Tokyo, Japan), 100 μg/ml streptomycin sulfate, and 10% heat-inactivated human AB serum (Biowest, Nuaillé, France). Cells (1 × 10⁵/well) were incubated in a total volume of 100 ml with the peptides or concanavalin A (4 μg/ml, Fujifilm Wako Pure Chemical Co., Osaka, Japan) in triplicates in 96-well round-bottom culture plates (Nunc, Roskilde, Denmark) at 37°C in a humidified atmosphere with 5% CO₂. The cultures were incubated for 4 days and then pulsed with bromodeoxyuridine (BrdU) during the last 16 h of incubation. Cellular proliferation was assessed using a BrdU enzyme-linked immunosorbent assay kit (Cell Proliferation ELISA BrdU, Sigma), according to the manufacturer’s instructions. After co-culture with Brdu for 16 h, the BrdU labeling solution was removed and anti-BrdU antibody was added. The OD at 450 nm for BrdU was measured using a luminescence plate reader (ARVO X3, Perkin Elmer Japan, Yokohama, Japan). The results were expressed as stimulation index (SI: mean OD_450nm in stimulated cultures / mean OD_{450 nm} in unstimulated control cultures).

HET, JTT, and NYT were purchased from Tsumura & Co. (TJ; Tokyo, Japan), Kracie Pharma Ltd. (KR; Tokyo, Japan), and Kotaro Pharmaceutical Co., Ltd. (KO; Osaka, Japan) (Table 1). HET, JTT, and NYT (10 mg) were dissolved in 2 mL of Roswell Park Memorial Institute- (RPMI-) 1640 medium containing 10% fetal bovine serum (FBS), centrifuged at 2190xg for 10 min at 23°C, and then passed through a 0.22 μm membrane filter (ADVANTEC MFS, Inc., Tokyo, Japan). The nine Hozai solutions were stored at −20°C until use. FBS and RPMI-1640 medium were purchased from Sigma-Aldrich (St. Louis, MO, USA). Penicillin-streptomycin (10,000 U/mL penicillin and 10,000 μg/mL streptomycin) and 10x phosphate-buffered saline (PBS) were purchased from Gibco (Grand Island, NY, USA). Concanavalin A was purchased from Wako Pure Chemical Corporation (Osaka, Japan), and Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies (Kumamoto, Japan). Paraformaldehyde was purchased from Thermo Fisher Scientific (Waltham, MA, USA). PerCP-Cy™5.5 mouse anti-human CD4, allophycocyanin (APC) mouse anti-human CD8, APC mouse anti-human CD25, Human Foxp3 Buffer A, Human Foxp3 Buffer B, phycoerythrin (PE) mouse anti-human Foxp3, BD Cytometric Bead Assay (CBA) Flex Set, and Human TGF-β Single Plex Flex Set were purchased from BD Biosciences (Franklin Lakes, NJ, USA).

Drosophila S2 cells were cultured in Schneider's Insect Medium (Gibco) supplemented with 10% FCS and antibiotics at 25°C (Schneider, 1972 (link)). pUAST vectors with actin5Ce-Gal4 drivers were cotransfected using Effectene (Qiagen) according to the manufacturer's instructions, and harvested 36-48 h after transfection. For immunofluorescence or time-lapse imaging, cells were replated on coverslips or glass-bottom dishes coated with Concanavalin A (Wako) and were allowed to spread for 1-2 h (Rogers et al., 2002 (link)). For drug treatments, cells were treated with 1 µM Latrunculin A (Wako) or 10 µM Colchicine (Wako) for 1 h before imaging. For immunofluorescence, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature, permeabilized with 0.1% Triton X-100 in PBS (PBS-T) for 15 min, and blocked with 5% skimmed milk in Tris-buffered saline (TBS). Primary and secondary antibodies were diluted in the blocking solution. After each antibody incubation, the coverslips were washed three times with PBS-T. The cells were mounted in Vectashield mounting medium (Vector Labs).