Ab3373

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.

Spheroids were collected, fixed with 4% formaldehyde for 24 h, cryoprotected in 30% sucrose and embedded in Tissue-Tek OCT compound (Sakura, The Netherlands). Spheroid cryosections (8 μm) were stained for CYP3A4 (PAP011, 1:5,000, Cypex Limited, United Kingdom), albumin (sc51515, 1:200, Santa Cruz, USA), E-cadherin (13–1700, 1:300, Thermo Scientific), MRP2 (ab3373, 1:100, Abcam, United Kingdom), CK19 (sc6278, 1:100, Santa Cruz), CD68 (sc70761, 1:50, Santa Cruz), Cleaved caspase-3 (9661, 1:500, Cell Signaling Technology, USA) and Vimentin (ab92547, 1:200, Abcam). CellProfiler software was used to quantify marker staining relative to spheroid size.

Immunoblotting and subsequent densitometry were performed with mixed plasma membranes, and intracellular membranes were incubated with antibodies against Mrp2 (ab3373, 1:50, abcam) and Na⁺/K⁺-ATPase (#3010, 1:1000, Cell Signaling). Moreover, the crude membranes were prepared according to the protocol of protein extraction kit (BioVision, USA). Equal amounts of crude membranes (40 μg/lane) were separated on 6%-15% SDS-PAGE and transferred to nitrocellulose membrane. Membranes were blocked in 5% skim milk and incubated with antibodies against Bsep (sc-25571, 1:200, Santa Cruz), Ntcp (#6522-1, 1:1000, epitomics), Oatp1 (LS-C113034-50, 1:1000, LifeSpan BioSciences), Mrp3(ab3375, 1:50, abcam), Mdr2 (SAB2100008, 1:1000, sigma) and Na⁺/K⁺-ATPase (#3010, 1:1000, Cell Signaling), respectively. Blots were incubated with horseradish peroxidase conjugated secondary antibodies (Santa Cruz) and developed by ECL detection regents (Amersham). The protein bands were quantified by the average ratios of integral optic density (IOD) following normalization to Na⁺/K⁺-ATPase expression.

The imHCs were cultured onto 96-well CellCarrier-96 optic black plates (PerkinElmer, Waltham, MA, USA) and stained with antibodies against hepatocyte markers: Albumin (ALB) (1:100 dilution, ab10241, Abcam), α-fetoprotein (AFP) (1:100 dilution, SC8399, Santa Cruz Biotech, Dallas, TX, USA), LDLR (1:100 dilution, SC373830, Santa Cruz Biotechnology), sodium taurocholate cotransporting polypeptide (NTCP) (1:100 dilution, ab131084, Abcam), MRP2 (1:100, AB3373, Abcam) and hepatocyte nuclear factor-4α (HNF-4α) (1:100 dilution, SC6556, Santa Cruz Biotech). For detecting HBV infectivity, infected hepatocytes were stained with antibodies against HBV proteins: HBcAg (1:100 dilution, ab8637, Abcam), HBsAg (1:100 dilution, ab20758, Abcam). Hepatocytes were then incubated with goat anti-mouse Alexa Fluor^® 488-conjugated (1:500 dilution, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), goat anti-rabbit Alexa Fluor^® 488-conjugated (1:500 dilution, Invitrogen), or donkey anti-goat Cy3-conjugated secondary antibody (1:500 dilution, BioLegend, San Diego, CA, USA). Hepatocyte nuclei were stained with 2 µM Hoechst 33342 (Thermo Fisher Scientific, MA). Mouse IgG2a, mouse IgG1, rabbit IgG and goat IgG were used as negative control for staining. Fluorescence images were captured by an Operetta High-Content Imaging System (PerkinElmer, MA) with a 40× objective lens.

Vessel was stopped for 60 minutes in order to harvest the cells inside the vessel in SMG condition.
In order to achieve semi-quantitative data, cells were removed from the vessel, were counted and, finally, were smeared on a slide to perform immunofluorescence analysis. Cells from NG and SMG conditions were fixed in pure acetone for 10 minutes at room temperature to be analyzed by immunofluorescence. Because of technical aspects, cell cultures did not retain their 3D organization, which did not reflect the appearance of the colony under microgravity. Primary antibodies against OCT4A (#2050; Cell Signaling), SOX2 (AB97959; Abcam), PDX1 (SC-25403; Santa Cruz), ALB (F0117; DAKO) and MRP2 (ab3373; Abcam) were used. Then, cells were washed and incubated for 1 hour with labeled isotype specific secondary antibodies (anti-mouse AlexaFluor-488, anti-rabbit Alexafluor-546, Invitrogen, Life Technologies Ltd, Paisley, UK) and 4,6′-diamidino-2-phenylindole (DAPI) was used to counterstain cell nuclei. Protein expression were examined by Leica Microsystems DM 4500 B Light and Fluorescence Microscopy (Weltzlar, Germany) equipped with a JenoptikProg Res C10 Plus Videocam (Jena, Germany) (Supplementary Table 2).