Dimethyl sulfoxide (dmso)

Naringenin (Tokyo Chemical Industry, Tokyo, Japan) was dissolved in dimethyl sulfoxide (DMSO; Fujifilm Wako Pure Chemical Corporation) at a concentration of 100 mg/mL. A corresponding concentration of DMSO was used for the control group.

Induction of metaxylem differentiation in cultured cells was performed as described previously (Oda et al., 2010 (link)). Briefly, 1 ml of 7-day-old suspension cells harboring LexA:VND6 was transferred into a 15-ml tube and diluted with 9 ml of MS medium without 2,4-dichlorophenoxyacetic acid. Cells were allowed to settle for 5 min, after which the upper 5 ml of the medium was removed to adjust cell density. The suspension culture was supplied with 2 μM estradiol (10 mM stock in DMSO, Fujifilm Wako) and 2 μM brassinolide (10 mM stock in DMSO, Fujifilm Wako), and cultured for 24 h. Transformation was performed as described previously (Oda et al., 2010 (link)). Seven-day-old cells were co-cultured with the Agrobacterium tumefaciens strain GV3101 MP90 (A gift from Dr Csaba Koncz, Max Plant Institute for Plant Breeding Research, Cologne, Germany) harboring LexA:ICR2-1×YPet and 35S:mScarleti-TUB6 for 48 h in MS medium supplemented with 50 mg/l of acetosyringone (Sigma-Aldrich). Claforan (0.5 mg/l; Aventis) was added to the culture, and the suspension cells were cultured for a further 5 days. Cell walls were labeled with WGA-Alexa Fluor 561 (Thermo Fisher Scientific).

5-FU (Kyowa Hakko, Tokyo, Japan) was administered to mice intraperitoneally (i.p.) at a dose of 150 mg/kg either as a single dose or once every 7 days two times. Recombinant Ang-1, Thpo and CXCL12 were purchased from R&D Systems (Minneapolis, MN) and diluted with phosphate-buffer saline (PBS). The pharmacological inhibitors PD98059 and LY294002 were purchased from Merck Millipore (Darmstadt, Germany) and diluted with dimethyl sulfoxide (DMSO; Wako, Osaka, Japan). The NF-κB inhibitor BAY-11-7082 (Wako) was reconstituted in DMSO (Wako) as a 10 mM stock solution and diluted with PBS. In animal experiments, BAY-11-7082 was administered i.p. at a dose of 10 mg/kg and mice were sacrificed after 12 h. The Sp1 inhibitor Mithramycin A (MIT) was purchased from Cayman Chemical (Ann Arbor, MI) and diluted with ethanol (Wako). Tolfenamic Acid (TA) was purchased from Wako and mixed with corn oil (Sigma, St. Louis, MO). MIT was administered i.p. once every 2 days, and TA was administered via oral gavage also once every 2 days. Verapamil was purchased from Eisai (Tokyo, Japan) and diluted with sterile water. LPS (Escherichia coli 055:B5) was purchased from FUJIFILM (Osaka, Japan) and diluted with PBS. LPS injections were administered intravenously (i.v.) at a dose of 250 µg/kg.

DCZ (HY-42110, MedChemExpress) was dissolved in dimethyl sulfoxide (DMSO, FUJIFILM Wako Pure Chemical Co.), then diluted in saline to a final volume of 1 mL (2% DMSO in saline), thus achieving 0.1 mg kg⁻¹ dose. Note that this dose of DCZ yields 80–90% hM4Di occupancy and affects behavioral performance via hM4Di activation in monkeys^{12 (link)}. Vehicle control was an i.m. injection of vehicle solution (2% DMSO in saline) at the same volume. Vehicle was delivered 15–20 min after delivery of the bicuculline. The first injection of DCZ was delivered 15–20 min after bicuculline (one case) or vehicle (5 cases). The second injection of DCZ was delivered 18–30 min after the first one (five cases). The timing of vehicle and DCZ administration for each session is summarized in Supplementary Table 1.

Ez was supplied by MSD, Tokyo, Japan. Ez was resolved in dimethyl sulfoxide (DMSO; Wako Pure Chemical Industry Ltd., Tokyo, Japan), and DMSO was used as a control. Antibodies used in this study were as follows: anti‐smooth muscle actin (Sigma, St. Louis, MO, USA), anti‐CD11b (Abcam, Cambridge, UK), and anti‐CD68 (KP1, Santa Cruz, CA, USA).