Polyoxyethylene octyl phenyl ether

ELISA was performed to analyze the levels of caspase-cleaved cytokeratin 18 (cCK18) using the M30-Apoptosense ELISA kit (Peviva Ab, Bromma, Sweden). Cells (5000 cells/well) were seeded in 96-well plates and treated with 10 μM lenvatinib or DMSO for 24 h. Subsequently, the cells were lysed in polyoxyethylene octyl phenyl ether (Wako) and analyzed according to the manufacturer’s instructions. The experiments were repeated three times.

The cultured cells were fixed and stained as previously described49 (link). Primary antibodies were as follows; mouse anti-human Hnf4a (PPMX), goat anti-human Foxa3 (Santa Cruz), goat anti-mouse albumin antibody (Bethyl Laboratories), mouse anti-mouse E-cadherin (Abcam), mouse anti-mouse cytchromeP450 1A2 (Abcam) at 1:200 respectively. The sections were incubated for two hours with secondary antibodies; Alexa 488-conjugated rabbit anti-goat IgG or goat anti-mouse IgG at 1:1000. After washing, the stained sections were covered with Vectashield mounting medium with DAPI (Vector Laboratories).
Spheroids were first embedded in iPGel (NIPPON Genetics) and fixed with 4% PFA overnight, then embedded in paraffin and cut in 5 μm thick. After deparaffinization and antigen retrieval using a Target Retrieval Solution (Dako), nonspecific binding was blocked with 1% bovine serum albumin (Sigma Aldrich) dissolved in 0.1% Polyoxyethylene Octylphenyl Ether (Wako) in PBS. The primary and secondary antibodies reactions were carried out in the same way for cultured cells.