Eclipseti microscopy

To certify the bone marrow endothelial cells for the expression of CD31 (PECAM-1), VE-cadherin (CD144) and ICAM-1 with passage zero were determined by direct immunofluorescence staining. The CD31 microbead-positive cells (50 000 cells) were plated into pre-coated 48-well with rat tail collagen type 1 for 5–7 days, as described above. After 60–70% of the confluence, the medium removed, cells fixed with 4% paraformaldehyde (PF) for 20 min at RT, followed by two wash 3 min apart with PBS. The cells were permeabilized with 100% cold methanol at room temperature for 20 min, rinsed with PBS three times, and blocked with 1% BSA/PBS for 1 h at room temperature. Cells incubated with the recommended dilution of primary antibodies overnight at 4°C. The cells were cleaned twice with PBS and counterstained with Hoechst for 5 min at 37°C. The cells were washed with PBS and imaging acquired using inverted Nikon microscopy (Nikon Eclipse Ti microscopy, Tokyo, Japan). Primary and secondary antibodies are shown in (Table 3) with a dilution of 1:100 for primary antibodies and 1:500 for secondary antibodies in 1%BSA/PBS solution.

Formalin-fixed liver cancer sections were treated with xylene and graded alcohols, followed by 15 min’s incubation in 3% Hydrogen Peroxide (H₂O₂). Then the sections were boiled 15 min in Tris/EDTA buffer for antigen retrieval. Then the samples were incubated in primary and secondary antibodies, followed by treatment with HRP substrate. Nikon-EclipseTi microscopy was used for observation.

HBx-GFP cell lines were transfected with shRNAs as indicated in the figure legends, and shRNA-expressing cells were selected in medium containing puromycin for 3 weeks. For cell proliferation assay, the cells were seeded in 24-well plates in triplicate at a density of 2000 cells per well in 0.5 ml of medium. Cell number at the indicated time points was determined by counting using a haemocytometer. Colony formation assay in soft agar was performed as previously described.^{37 (link)} Briefly, 5 × 10³ cells per well were resuspended in DMEM containing 0.3% agar. Then, the suspension was laid over DMEM containing 0.5% agar in each of the triplicate wells of a 6-well plate. The plates were incubated for 14 days in a 5% CO₂ incubator at 37 °C, with replenishment of medium every 3 days. Colonies were imaged using Nikon ECLIPSE TI microscopy and colonies in three randomly chosen fields per well were counted for quantification.

Cultured cells were grown on coverslips placed in a six-well plate. Forty-eight hours after transfection, cells were subsequently fixed by 4% paraformaldehyde and permeabilized by Triton X-100. After blockage, cells were incubated with primary antibodies at 4℃ overnight and secondary antibodies with fluorophore label at room temperature for 1 hr, in moist environmental box. At last, Hoechst 33342 was added to stain nucleus of cells. Cell images were captured using a Nikon ECLIPSE Ti microscopy.

Alkaline phosphatase (ALP) staining was performed using the Alkaline Phosphatase Detection Kit (#SCR004, Millipore Inc., USA). Briefly, reprogrammed cells were fixed with 4% paraformaldehyde/PBS for 2 min, followed by 15 min incubation with staining solution according to the manufacturer's protocol. For immunocytochemistry, cells were fixed in 4% paraformaldehyde/PBS for 20 min at room temperature. The cells were then permeabilized with 70% ethanol and stored at 4°C. After washing with PBS, cells were blocked with 10% BSA/PBS for 2 h at room temperature. Slides were incubated with primary antibodies in 10% BSA/PBS for 2 h at room temperature or overnight at 4°C, washed three times with PBS and incubated with the corresponding secondary antibodies conjugated with Dylight variants. Following incubation, cells were washed three times with PBS and nuclei stained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma Inc., USA). The slides were mounted with mounting medium, Vectashield (Vector Inc., USA) and cells were visualized using confocal microscopy (Nikon Eclipse Ti microscopy). The antibodies with their sources and dilutions are listed in Table S5.