Sr b1

For Western blotting, cells were lysed using RIPA buffer. Equal amounts of protein (30 μg) were loaded and separated by 8% SDS-PAGE. Separated proteins were electrophoretically transferred to a nitrocellulose membrane, blocked with 5% (w v^− 1) skim milk solution for 1 h, and then incubated with primary antibodies to CD36 (Santa Cruz Biotechnology, Inc., CA, USA; Cat. No. sc-9154), SR-B1 (Abcam, Inc.; Cat. No. ab-106,572), ABCA1 (Abcam, Inc.; Cat. No. ab-18,180) and GAPDH (Abcam, Inc.; Cat. No. ab-181,602), respectively, overnight at 4 °C. Blots were visualized by an ECL system (Pierce).

Immunofluorescence of HepG2 cells was performed by following the previously described protocol [17 (link)]. Cells were fixed in paraformaldehyde (4% in PBS) and incubated overnight with appropriate antibodies: HMGCR (Abcam, ab242315, dilution 1:100), SREBP-2 (Abcam, ab30682, dilution 1:100), SREBP-1 (Santa Cruz Biotechnology, Dallas, TX, USA, sc-8984, dilution 1:100), SR-B1 (Abcam, ab52629, dilution 1:100), LDLr (Santa Cruz Biotechnology, sc-11824, dilution 1:200), anti-Perilipin-2 (anti-Plin2) antibody (R&D Systems, #MAB76341, dilution 1:100). After incubation with primary antibodies, fixed cells were probed for 1 hour at room temperature with donkey anti-goat secondary antibody Alexa Fluor 488 (ThermoFisher Scientific, Milan, Italy, A-11055), goat anti-rabbit secondary antibody Alexa Fluor 555 (ThermoFisher Scientific, A27039) and goat anti-rabbit secondary antibody Alexa Fluor 488 (ThermoFisher Scientific, A-11008). Coverslips were mounted with Vectashield Antifade mounting medium with DAPI (Vector, H-1200) to visualize nuclear staining. The samples were examined at confocal microscopy (TCS SP8; Leica, Wetzlar, Germany). Images were captured using Leica TCS SP8 equipped with a 40 × 1.40–0.60 NA HCX Plan Apo oil BL objective at RT and Leica LAS X Software.

Human liver tissue arrays were purchased from US Biomax (Derwood, MD). ASGR staining IHC was performed as previously described.³⁹ The ASGR staining was reviewed by the pathologist (S.K.) and quantified in H-scores using the following equation: H-score = (“3+” % cells) *3 + (“2+” % cells)*2 + (“1+” % cells)*1 + (0% cells)*0, where “0” means no staining; and “1+, 2+, and +3” mean weak, moderate, and strong staining, respectively. Samples containing no tumor cells or of mixed HCC-cholangiocarcinoma (CCA) or with unknown tumor grade were excluded from the calculation of H-scores. Antibodies specific for ASGR (Proteintech, Rosemont, IL), cleaved active caspase-3 (Cell Signaling Technology, Danvers, MA), SR-B1 (Abcam, Cambridge, MA), and ASO were used at 1:100, 1:400, and 1:10,000 dilutions, respectively. The antibody specific for MyD88 (Novus Biologicals, Littleton, CO) was used at 2.5 μg/mL. For fluorescent IHC, spheroids were fixed in 4% paraformaldehyde and incubated with mouse anti-ASGR antibody (Santa Cruz Biotechnology, Dallas, TX) at 4°C overnight, followed by staining with Cy3-conjugated goat anti-mouse immunoglobulin G (IgG) antibody (Beyotime, Haimen, China) and co-staining with DAPI at room temperature for 30 min.