Staurosporine

iHEPs were treated +/−20 nM staurosporine (Generon) for 2 h, after which the cells were fixed in 4% PFA (Sigma-Aldrich) in PBS for 15 min. The fixed cells were then blocked for 1 h in blocking buffer (0.2% Triton X-100 (Sigma-Aldrich), 3% Donkey-Serum (Sigma-Aldrich) and 1% BSA (Sigma-Aldrich) in PBS) at room temperature. Next, the cells were stained with a 1:25 dilution of human MRP2 primary antibody (Abcam; ab3373) in blocking buffer overnight at 4 °C. The following day, the cells were stained with a 1:500 dilution of AlexaFluor 555 donkey anti-mouse antibody (Invitrogen; A-31570) in PBS for 2 h at room temperature, followed by a 5-min incubation with NucBlue Fixed Cell ReadyProbes Reagent (DAPI; Thermo Fisher Scientific) at room temperature. Cell nuclei (blue) and MRP2 expression at the canalicular membrane (red) were captured in images taken on an LSM 880 confocal microscope (ZEISS). Images were analysed using Fiji^{45 (link)} as above to determine integrated density of MRP2 staining at the canalicular membrane, total canalicular area, average canalicular area and canalicular count. The statistical significance of experimental data was assessed by Student’s test.

Human iPSCs (CGT-RCiB10, Cell and Gene Therapy Catapult) were differentiated into iHEPs essentially as described^{44 (link)}, except for the Matrigel (Corning) sandwich and addition of forskolin (5 μM; BioGems) from day 17. On day 19 hepatocyte growth factor was removed and taurine was added (58.4 mg/L; Sigma) and the cells are placed in normoxia. On day 25 the iHEPs were treated +/−20 nM staurosporine (Generon) for 2 h followed by addition of the MRP2 transport substrate precursor CDFDA (10 µM; Sigma-Aldrich) and NucBlue Live Cell ReadyProbes Reagent (Thermo Fisher Scientific) and incubated for a further 15 min at 37 °C. CDFDA is cleaved by intracellular esterases to yield fluorescent 5-(and-6)-carboxy-2′,7′–dichlorofluorescein (CDF; green) which is secreted into the bile canaliculi by MRP2. Cell nuclei and accumulation of CDF (green) in bile canaliculi were captured in images taken on an LSM 880 confocal microscope (ZEISS). Images were analysed using Fiji^{45 (link)}. Images were first processed using global thresholding to filter out noise. Then, size-exclusion filtering was applied to remove particles <5 µm² to select biologically relevant signals to determine integrated density of CDF staining in the bile canaliculi. The statistical significance of experimental data was assessed by Student’s test.