Sc 6556

Antibody staining and TUNEL assay were performed as described previously (29 (link)). The following antibodies were used: antibodies against Dendra2 (1:1000; AB821, Evrogen, Moscow, Russia), phospho-4E-BP1 (Thr-37/46) (1:500; catalog no. 2855, Cell Signaling), Hnf4a (1:200; sc-6556, Santa Cruz Biotechnology, Inc.), Prox1 (1:500; ab5475, Chemicon), 2F11 (1:1000; ab71826, Abcam, Cambridge, MA), and PCNA (1:1000; SAB2701819, Sigma).

Protein from whole cell lysates was run on 4% to 12% Tris-Bis acrylamide gels (NP0321, Thermo Fisher Scientific, Waltham, MO, USA) using the NuPAGE system (Thermo Fisher Scientific, Waltham, MO, USA). Protein was transferred to PVDF blotting membranes (1620177, Bio-Rad, Hercules, CA 94547) using wet electroblot apparatus. Blots were blocked with 5% non-fat milk in TBST. Primary antibodies (HNF4A, 1:500, sc-6556; Santa Cruz Biotechnology, Santa Cruz, CA, USA and GAPDH, 1:1000, DB600-502; Novus Biologicals, Centennial, CO, USA) and secondary antibodies (Donkey anti-goat IgG HRP and Donkey anti-mouse IgG HRP) were applied to the blots in 5% non-fat milk in TBST. Three 5 min TBST washes were performed after each antibody incubation. Following the last wash, SuperSignal West Pico Chemiluminescent substrate (34080, Thermo Fisher Scientific) was applied to the blots for 3 min. The blots were then exposed to films (F-9023, GeneMate, VWR, Radnor, PA, USA), which were developed in an SRX-101A developer (Konica Minolta, Ramsey, NJ, USA).

For immunofluorescence labeling, liver sections were (co-)stained with antibodies directed against GS (1:1000; ab73593, Abcam), SOX9 (1/100; AB5535, Millipore), F4/80 (1:100; MCA497RT, BioRad), HNF4ɑ (1/100; sc6556, Santa Cruz) or CD31 (1:200; MEC13.3, BioLegend). Secondary antibodies were either anti-rabbit AlexaFluor 488; or anti-rabbit, anti-goat or anti-rat AlexaFluor 647 (1:1000, Invitrogen). Hoechst (1:10 000, 62249, Thermo Scientific) was used to counterstain the nuclei. Liver sections were imaged using the Zeiss AxioImager.Z2 microscope.

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.

The antibody to NBCe1 (Sigma WH0008671M1) has been well validated in human renal tissue in our previous studies in the RPT [17 (link)]. The NBCe2 antibody (Sigma HPA036621) used previously [17 (link)] and in the current studies was characterized in the Human Protein Atlas (http://www.proteinatlas.org/ENSG00000188687-SLC4A5/tissue/kidney#imid_20081509). We further verified its specificity using confocal imaging with dual staining for hRPTC-specific marker CD-13 (BD 347837, 1:500 dilution) or HNF4A (goat polyclonal anti-HNF4A, Santa Cruz sc-6556, 1:500). This was performed in cultured primary and immortalized hRPTCs, including overexpressed cell lines or those stably transfected with NBCe2-specific siRNA to knockdown SLC4A5, the NBCe2 gene.