T75 flask

Retroviruses (carrying the hTERT gene and those carrying activated ras gene) were generated using the Phoenix A packaging cell line (kind gift from the Nolan laboratory). The day before transfection, 4 million Phoenix A cells were seeded in a T-75 flask (Sarstedt, 83.3911.002) in DMEM (Sigma-Aldrich, D6429) supplemented with fetal calf serum (as above) up to 10%. The next day, 10 µg pBabePurohTERT (Addgene) or pBabePuroRasV16 (kind gift from R. Weinberg) were mixed with calcium phosphate reagents, which were procured from Promega (Profection Mammalian Transfection System, Promega, E1200) according to the accompanying instructions. The next day, the media of the cells were removed, the monolayer of cells rinsed once with HBSS (as above) and replaced with media of the target cells (as above). The next day the virus-containing media were collected and filtered through a 0.2-µm filter. Polybrene (Millipore, TR-1003-G) was added up to 8 µg ml⁻¹, and the virus-Polybrene mixture was added to the target cells. The next day, the media were replaced with fresh media containing 1 µg ml⁻¹ puromycin (Invitrogen, A11138–03). Antibiotic selection continued until all uninfected target cells, which were also similarly processed, were dead.

Stable HEK293 cell lines were generated as previously described^{38 (link)}. In brief, MSCV retrovirus particles were produced by co-transfecting PlatE cells with the cloned vector and a GagPol helper plasmid using calcium phosphate transfection. Forty-eight hours after transfection, the supernatant containing the retrovirus particles was harvested and filtered through a 0.45 µm syringe filter. Next, 3 × 10⁵ HEK293 target cells (expressing EcoR) were seeded into the well of a six-well plate, and the virus supernatant was added dropwise. Forty-eight hours post infection, antibiotic selection (300 µg/mL neomycin or 200 µg/mL hygromycin) was initiated. After the selection process, the infected cells were expanded, and either used directly for cell culture experiments, or cryopreserved at -80 °C upon later usage. For the simultaneous expression of two N6-MTases and in the GFP-reporter assay, stable double cell lines were generated. For this, the previously established single cells lines were re-infected with a second construct using the same workflow as described above. In each case, the infected and selected cells were transferred into a T-75 flask (Sarstedt) and expanded. A first fluorescence activated cell sorting (FACS) step was performed as described in the next paragraph in order to remove “leaky” cells expressing the constructs without doxycycline (dox) treatment.

FAM-scr-GreenB1 and FAM-GreenB1 were folded at 1 μM in binding buffer and diluted (500, 250, 125, 25, and 5 nM). MCF-7, MDA-MB-231, and MDA-MB-436 cells were cultivated in a T75 flask (Sarstedt) until 80% confluence. Cells were washed with PBS and dissociated using non-enzymatic cell-dissociation buffer (25-056-CI, Corning) for 5–9 min, followed by addition of complete culture medium, centrifugation at 300 × g for 5 min and removal of the supernatant. Cells were washed twice with binding buffer, split into samples, and resuspended with different concentrations of FAM-scr-GreenB1 or FAM-GreenB1 (n = 3 for each concentration, aptamer and cell line). Samples were incubated on ice for 1 h, washed twice with washing buffer, resuspended in 40 μL of binding buffer, and analyzed using an Amnis ImageStream^X Mk II imaging flow cytometer and IDEAS software (Luminex) or an Accuri C6 Plus (BD Biosciences) flow cytometer. Statistical significance was determined using unpaired t tests, and statistical significance was adjusted for multiple comparisons using the Holm-Šídák method. Apparent K_D was calculated by subtracting the FAM-scr-GreenB1 non-specific signal from the FAM-GreenB1 signal, and data were fitted using one site-specific binding model. All calculations were done using Prism 9.3.1 (GraphPad).

Mononuclear cells from BM aspirates were cultured at a density of 2 × 10⁶ cells in a T75 flask (Sarstedt, Germany) in DMEM with 1 g/l glucose, 4 mM glutamine, and 1 mM sodium pyruvate (all from Life Technologies, Germany), supplemented with 10% MSC-qualified FBS (WKS Diagnostik, Germany), 1% penicillin/streptomycin (Life Technologies, Germany), and 1 ng/ml bFGF (Peprotech, Germany). Adherent cells were cultured for 3 weeks and cryo-conserved. Before cryo-conservation MSCs were tested for the expression of CD45, CD34, CD90, CD105, CD44, and Nestin by flow cytometry (Supplementary Fig. 4). In addition, the ability of MSCs to differentiate into adipocytes and OBs were tested^{71 (link)}: OBs for coculture experiments were generated by culturing confluent MSC cultures in DMEM with high glucose (Life Technologies, Germany) supplemented with 10% MSC-qualified FBS (WKS Diagnostik, Frankfurt, Germany) 1% penicillin/streptomycin (Life Technologies, Germany), 10⁻⁷ M dexamethasone, 25 µg/ml l-ascorbic acid, and 3 mM sodium dihydrogen phosphate (all from Sigma-Aldrich, Germany) for 21 days with the medium being changed every other day.

A549 cells were maintained inside a T75 flask (Sarstedt, Germany) in a cell culture incubator (37 °C, 5% CO₂). Cells were subcultured every 3–4 days, when the cells reached 80% confluence. For QuasiVivo experiments, coverslips were sterilized with 70% ethanol and inserted into a 24-well plate. After ethanol had evaporated, each well was washed twice with sterile 1 × Dulbecco’s phosphate buffered saline without magnesium and calcium (DPBS) (Gibco™, United Kingdom). Then, 100,000 cells/well were seeded on each coverslip. Cells were let to attach for either 24 or 48 h depending on the experiment.

Two RVFV strains were selected for this study: the wt ZH548 strain and a recombinant RVFV virus derived from the ZH548 strain by substituting the NSs protein with the far-red fluorescent protein Katushka, hereafter termed ΔNSs::Katushka [37 (link)]. The viral genomic organisation of both strains is presented schematically in Figure 1.
For viral propagation, Vero B4 cells [38 (link)] were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Thermo Scientific) supplemented with 5% FBS and 0.2% PE/ST. One day before viral inoculation, 2 × 10⁶ Vero B4 cells were seeded in a T75 flask (Sarstedt, Nümbrecht, Germany). Next, cells were infected with RVFV at a multiplicity of infection (MOI) of 0.01 for 1 h and maintained in DMEM with a lower FBS concentration (1%). The supernatant containing virions was collected 72 h post-infection and titrated by using a plaque assay. All work involving the wt ZH548 strain was performed under biosafety laboratory 3 (BSL-3) conditions; the ΔNSs::Katushka strain used in the study was handled in BSL-2 conditions, as permitted by the Swedish Work Environment Authority.

Normal human dermal fibroblasts (NHDF) (PromoCell, C12300) were cultured in Quantum 333 (PAA, U15-813) supplemented with 1% (v/v) penicillin/streptomycin (PAA, P11–010). NHDF are human primary cells which were isolated from the dermis of juvenile foreskin or adult skin with different origins (PromoCell). For stock cultures, 750000 cells in 20 ml medium were seeded weekly into a T75 flask (Sarstedt) and kept at 37°C and 5% CO₂ in a humidified atmosphere. The medium was changed every two to three days. For sub-culturing, cells were trypsinized by Trypsin/EDTA (PAA, L11-659). Cells were counted under the microscope.