395b 1kt

PAS and CD31 detection in FFPE slides was conducted as previously described [54 (link),55 (link)]. The slides were first immunostained with CD31 (Abcam, Cambridge, UK, ab124432) as described above. After secondary antibody incubation, PAS staining was performed following the manufacturer’s recommendations (Sigma, St. Louis, MO, USA, 395B-1KT). Briefly, the slides were incubated for 5 min at room temperature with PAS, washed, and incubated for 15 min with Schiff’s Reagent. The slides were then counterstained with hematoxylin, dehydrated, and mounted.

For growth assays, 50 × 10⁵ cells were seeded and grown in DMEM containing glucose (4.5 g l⁻¹) or glucose-free DMEM supplemented with galactose (4.5 g l⁻¹). Cells were counted manually on a haematocytometer for 5 days. For metabolite concentration assays, TKO HCC cells were infected with either empty or Rb7LP-MigR1-IRES-GFP. Cells were collected 24 h after infection and sorted for GFP expression on a MoFloXDP sorter (Beckman-Coulter). The GFP⁺ fraction was replated and cultured for 48 or 72 h. Media were collected from cell cultures and assayed using an YSI 7100 reader (equipment generously provided by Kathryn Wellen, University of Pennsylvania). Reactive oxygen species (ROS) production was assayed using MitoSOX Red Mitochondrial Superoxide Indicator (Invitrogen, M36008). TKO cells infected with Rb7LP-MigR1 or empty MigR1 were harvested after 48 or 72 h and stained with Mitosox according to the manufacturer's specifications. Cells were analysed on a C6 Accuri analyzer (BD). For paraffin sections, organs were fixed in 4% formaldehyde overnight at room temperature and transferred to 70% ethanol before processing and embedding. Periodic acid–Schiff base staining was performed according to the manufacturer's specifications (Sigma, 395B-1KT). Slides were counter stained with haematoxylin and eosin where indicated. Slides were imaged on an Olympus IX81 inverted microscope.

Cytopins were prepared, according to the manufacturer´s instructions, utilizing the Cytospin 2 centrifuge (Thermo-Shandon, Pittsburgh, PA, USA). Shortly, 10.000 CD34+ cells were resuspended in 100 µl PBS and pipetted inside of a special plastic chamber connected with a special metal clip to a glass slide and a filter card (Thermo-Shandon, Pittsburgh, PA, USA) and centrifuged at 700 rpm for 5 min.
The generated cytospins were stained using the Periodic acid Schiff (PAS) method (395B-1KT, Sigma-Aldrich, St Louis, MO), for detection of the glycogen storage in each cell, according to the manufacturer’s instructions. Shortly, cells were fixed with 4% paraformaldehyde, incubated with 0.5% periodic acid solution for 5 min, and stained with Schiff's reagent for 15 min. This is followed by counterstaining with hematoxylin solution for 1.5 min. All steps were performed at room temperature, and cells were rinsed with tap water after each step^{30 (link)}.