Hepa1 6 cells

HEPA 1-6 cells (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum, 50 U/mL penicillin, and 50 µg/mL streptomycin in humified atmosphere at 37 °C and 5% CO₂. Syngeneic Hepa 1-6 tumor cells (5 × 10⁶) were subcutaneously injected into the flank of Dhx15^+/− and wild-type mice (n = 10). Primary tumor growth was controlled during the first 5 weeks. Tumor growth was monitored by measuring volumes using a digital slide-caliper. Tumor volume was calculated by the following formula: V = 4/3 × π × (length × depth × width). Primary tumors were fixed in 4% PFA and cryopreserved in tissue-tek O.C.T. compound (Sakura, Flemingweg, The Netherlands). The post-surgical metastasis model was performed as follows: five weeks post-injection, Hepa 1-6-injected mice were sacrificed, and the liver was extracted to perform metastasis analyses. Tile scan images of hematoxylin–eosin (H&E)-stained paraffin liver sections were visualized using an Olympus BX51 microscope equipped with DP71 camera (Olympus Europa SE & CO.KG., Hamburg, Germany), and the percentage of hepatic metastatic area as percent of total hepatic area was measured with ImageJ software (ImageJ version 1.52b; National Institutes of Health, Bethesda, MD, USA).

P. berghei and P. yoelii blood stage parasites were propagated in female Swiss mice (6–8 weeks old, from Janvier Labs). We used wild type P. berghei (ANKA strain, clone 15cy1) and P. yoelii (17XNL strain, clone 1.1), and GFP-expressing PyGFP and PbGFP parasite lines, obtained after integration of a GFP expression cassette at the dispensable p230p locus.²⁵ (link)
Anopheles stephensi mosquitoes were fed on P. berghei or P. yoelii-infected mice using standard methods,⁷² (link) and kept at 21°C and 24°C, respectively. P. berghei and P. yoelii sporozoites were collected from the salivary glands of infected mosquitoes 21–28 or 14–18 days post-feeding, respectively. P. berghei and P. yoelii sporozoite infections were performed in female C57BL/6 or BALB/c mice, respectively (6 weeks old, from Janvier Labs), by intravenous injection in a tail vein. HepG2 (ATCC HB-8065), HepG2/CD81³⁸ (link) and Hepa1-6 cells (ATCC CRL-1830) were cultured at 37°C under 5% CO₂ in DMEM supplemented with 10% fetal calf serum and antibiotics (Life Technologies), as described.⁷ (link) HepG2 and HepG2/CD81 were cultured in culture dishes coated with rat tail collagen I (Becton-Dickinson).

HeLa cells and Hepa 1-6 cells were purchased from ATCC. Cells were cultured at 37°C under a 5% CO₂ atmosphere in DMEM (containing 4.5 g/L of glucose) supplemented with 10% fetal bovine serum (Thermo Fischer Scientific), 100 IU/mL of penicillin G, and 100 μg/mL of streptomycin sulfate. Unless otherwise specified, cells were seeded at a density of between 6 × 10³ and 1 × 10⁴ cells per well in a 96-well plate, depending on the experiment.