R verapamil

The A.cinnamomea was a kindly gift from Cosmox Biomedical Co., Ltd. (Taoyuan, Taiwan). The preparation of the ethanolic extracts of fruiting bodies of A.cinnamomea (EEAC), methods of HPLC and MS analysis were the same as our previous studies [14] (link), [15] (link) and the results were shown in Figure S1. Rhodamine 123, calcein-AM, and R-(+)-Verapamil were purchased from Sigma Chemical Co (St. Louis, MO, U.S.A.). All restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA). The Flp-In ™ system, Flp-In™-293 cells (human embryonic kidney cells), zeocin, hygromycin B and all cell culture medium and reagents were obtained from Invitrogen (Carlsbad, CA, USA). Human ABCB1cDNA in pMDRA1 was provided by the Riken BRC DNA bank (RDB No. 1372) (Ibaraki, Japan).

MDR1 transporter activity in hiHepPC spheroids was analyzed as described previously^{33 (link)}. Briefly, 3D cultures of hiHepPC spheroids were incubated with 100 µM rhodamine 123 (Sigma-Aldrich) for 10 min at 37 °C under 5% CO₂. For inhibition of MDR1 transporter activity, rhodamine 123 was added to the culture medium after 30 min of incubation with 20 µM R(+)-verapamil (Sigma-Aldrich). The function of CFTR in the hiHepPC spheroids was evaluated as described previously^{33 (link)}. Briefly, hiHepPC spheroids were cultured in human cholangiocyte culture medium without FSK for 24 h, and then 10 µM FSK or FSK plus 100 µM CFTRinh-172 (Sigma-Aldrich) were added to the medium. The diameter of 20 spheroids was measured before addition of FSK or FSK plus CFTRinh-172 to the medium, and the diameter of corresponding spheroids was also measured after culture with or without FSK and/or CFTRinh-172 for 24 h.