Mts kit

The MTS kit (ab197010, Abcam, Chicago, IL, USA) and CCK‐8 kit (C0037, Beyotime Biotechnology, Shanghai, China) were used the cell proliferation assay. The detail of procedure was described previously [17 (link)].

H460-R cells were seeded in a 96-well plate at 7000 cells/well for 12–16 h. Next, cells were treated with cis-diamminedichloroplatinum (DDP) at 0, 2, 4, 8, and 16 μg/mL for 72 h, respectively. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium, inner salt (MTS) kit (Abcam Inc.).

Mouse cholangiocytes were plated in six-well plates (for ELISA assays) or in chamber slides (for IF assays) at 30–40% confluency and transfected the same day using Lipofectamine 2000 according to manufacturer’s instructions. The GHS-R1a and negative control siRNAs were purchased from Ambion through Thermofisher (Waltham, MA). Validation for GHS-R1a knockdown was performed in cholangiocytes 48 h after the siRNA transfection, using RT² qPCR Primer Assays for GHS-R1a purchased from Qiagen (Frederik, MD). Also, at 48 h after the transfection, the cells were treated with vehicle or Ghr for 30 min, and then processed for ELISA or IF and confocal microscopy. Cell proliferation of transfected cells was also assessed using the MTS kit from Abcam. In this experiment the cells were plated in 96-well plate with transfection reagents, incubated for 48 h, followed by treatments with vehicle or Ghr in the presence of MTS reagent for 4 h.