Ab131084

HB (collection in Sichuan, 20042901), ZZ (collection in Fujian, 210303), DH (collection in Gansu, 20201211), and MX (collection in Jiangsu, 21032501) were obtained from Simcare Ltd. Primary antibodies against ZO-1 (21773-1-AP, 1:1000), occludin (13409-1-AP, 1:1000), claudin-1 (13050-1-AP, 1:1000), and the second antibody of beta-actin (20536-1-AP, 1:10000) were purchased from Proteintech Group Inc. Primary antibodies against NTCP (ab131084, 1:1000), BSEP (ab155421, 1:1000), CYP7A1 (ab234982, 1:1000), MRP2 (ab172630, 1:1000), and FXR1 (ab129089, 1:1000) were obtained from Abcam Inc. ANIT (N106389), UDCA(U110695), and olive oil (O108685) were purchased from the Aladdin Chemical Reagent Co. Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (reference D4540) (St-Louis, MO, USA). The TRIzol total RNA extraction kit was obtained from Life Technologies.

The protein extraction protocol from cancer cells was previously reported [30 (link)]. Briefly, cells were collected and lysed using Radio Immunoprecipitation Assay (RIPA) protein extraction reagent (Beyotime) with a protease inhibitor cocktail (Roche, IN, USA). Equal amounts of protein lysate were quantified using BCA kit (Thermo Fisher, USA), separated by 10% sodium dodecyl sulfate–polyacrylamide gel (SDS–PAGE) electrophoresis, and transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was then sealed with 5% non-fat milk for 1 hour, and incubated with primary antibodies (Abcam, Cambridge, UK) against: FUS (ab84078, 1/1000), SLC10A1 (ab131084, 1/1000), β-actin (ab8227, 1/1000), GLUT1 (ab14683, 1/2500), HK2 (ab227198, 1/5000), LDHA (ab47010, 1/1000), PGK1 (ab154613, 1/1000) overnight at 4°C, with GAPDH (ab9485, 1/2500) as control. After washing, the membrane was incubated with goat anti-rabbit secondary antibody (ab96899, 1/1000, Abcam, Cambridge, UK) at 37°C for 1 hour. The protein bands were finally detected using enhanced chemiluminescence reagents (Bio-Rad, Hercules, CA, USA) according to manufacturer's instructions.

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.