Heparin

p19^ARF-/- and wt mLSECs were cultivated on collagen-coated (Collagen Type I-Rat Tail, BD Biosciences, USA, Cat.#354236) Petri dishes in DMEM (Dulbecco´s Modified Eagle´s Medium) containing 100 μg/ml Endothelial Cell Growth Supplement (ECGS, Biomedical Technologies, USA, Cat.#BT203), 0.2 μg/ml hydrocortisone (Alfa/Aesar, Germany, Cat.#A16292) and 50 μg/ml heparin (AppliChem, Germany, #3U009511). Human hepatic sinusoidal endothelial cells (hHSECs) (ScienCell, USA, Cat#5000) were cultivated in Endothelial Cell Medium (ECM) (ScienCell, USA, Cat. #1001) following the manufacturer’s instruction. Human telomerase reverse transcriptase (TERT)-immortalized blood endothelial cells (BECs; [27 (link)] were cultivated on collagen-coated plates in ECM. MIM1-4 cells were cultivated as described previously [25 (link)]. All cell lines were cultured at 37°C and 5% CO₂ and routinely screened for the absence of mycoplasma.

CHO-K1 and glycosaminoglycan (GAG)-deficient pgsA-745 cells seeded in 12-well plates were infected with virus at an MOI of 0.0001 in PBSA for 1 h at room temperature. Cell monolayers then were washed twice with PBS and overlaid with an agarose–GMEM overlay supplemented with 2% FCS. After 3 days at 37°C, cells were fixed and titers calculated as described for other plaque assays.
To remove heparan sulfate from the surface of BHK-21 cells, cells were seeded in 12-well plates and then incubated with 0.5 U/ml of heparinase I (Sigma) or BSA as a control for 30 min at room temperature with constant agitation. Subsequently, the mixture was replaced with virus in PBSA and incubated for a further 30 min at room temperature with constant agitation. Monolayers then were washed twice with PBS and overlaid with an agarose–GMEM overlay supplemented with 2% FCS. After 2 days at 37°C, cells were fixed and titers determined as described for plaque assays.
For heparin competition assays, virus was incubated with 200 μg/ml of heparin (AppliChem) or BSA as a control for 30 min on ice with constant rotation. BHK-21 cells were exposed to the virus-heparin mix for 30 min on ice, the inoculum was removed, the monolayers were washed twice with PBS, and an agarose–GMEM overlay supplemented with 2% FCS was added. After 2 days at 37°C, cells were fixed and titers determined as described for plaque assays.

The murine p19^ARF-deficient, EMT-transformed MIM-RT hepatocytes expressing green fluorescent protein (GFP) were cultivated in RPMI 1640 plus 10% fetal calf serum (FCS) and continuously supplied with 1 ng/mL TGF-β1 (Peprotech, Rocky Hill, NJ, USA) as described previously [25 (link)]. The murine p19^ARF-deficient liver sinusoidal endothelial cells, termed mLSECs, were propagated on collagen-coated (Collagen Type I-Rat Tail, BD Biosciences, San Jose, CA, USA, Cat.#354236) petri dishes in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 100 µg/mL Endothelial Cell Growth Supplement (ECGS; Biomedical Technologies, Stoughton, MA, USA, Cat.#BT203), 0.2 µg/mL hydrocortisone (Alfa/Aesar, Karlsruhe, Germany, Cat.#A16292), and 50 µg/mL heparin (AppliChem, Darmstadt, Germany, #3U009511) as outlined recently [27 (link)]. For fluorescent labelling, mLSECs cells were lentivirally transmitted with a vector harbouring red fluorescent protein (RFP), resulting in mLSECs-R cells. Human umbilical vein endothelial cells (HUVECs) were grown in Endothelial Cell Medium (ECM; ScienCell, Carlsbad, CA, USA, Cat.#1001) following the manufacturer’s instruction. All cells were grown at 37 °C and 5% CO₂, and were routinely screened for the absence of mycoplasma.