Six well tissue culture plates

Electroporation of full-length KRas constructs (KRasWT, KRasG13C, KRasG13C-edaGDP, KRasG13C:acetyledaGDP and KRasG13C-edaGppCp) was performed based on the protocol described by Alex et al., 2019 (link) using the Neon Transfection System Kit (Thermo Fisher). For electroporation, 3 million cells per experiment were harvested by trypsinization, washed with PBS, and resuspended in 85 µL of the electroporation buffer R (Thermo Fisher). Increasing amounts of recombinant protein samples for each construct (0, 50, 100, 200 and 300 µg) were diluted 1:1 in buffer R followed by the addition of 30 µL of this protein master mix to the cell suspension. This cellular slurry was loaded into a 100 mL Neon Pipette Tip (Thermo Fisher) and electroporated with 2x35 ms pulses at 1000 V. After electroporation, the cells were washed twice with PBS (15 mL) to remove non-internalized extracellular protein and the cell pellet was resuspended in 2 mL complete growth media. Cells were transferred into six-well tissue culture plates (Sarstedt) and incubated for 24 hr at 37 °C and 5% CO₂ in a humidified incubator for recovery before being processed for western blotting analysis.

MEFs were seeded at 1 × 10⁵ cells per well in six-well tissue culture plates (Sarstedt). At confluency, cells were induced into adipogenesis by incubating with differentiation media (DM) containing DMEM and 10% FBS supplemented with 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich), insulin (10 μg/ml; Sigma-Aldrich), 1 μM dexamethasone (Sigma-Aldrich), and 10 μM rosiglitazone (Sigma-Aldrich). Cells were treated for 3 days (D3), followed by replacement of DM with DMEM, and 10% FBS, and incubation for another 6 days (D9). MEFs were washed with 1 ml of 1× PBS (Sigma-Aldrich) once before fixing for 5 min with 4% paraformaldehyde in PBS at room temperature. After fixation, MEFs were washed three times with PBS and once with 60% isopropanol and were completely air-dried. Triglyceride accumulation was unveiled by staining with Oil Red O (ORO; Sigma-Aldrich) for 10 min at room temperature. Excess stain residue was removed with four ddH₂O washes. Approximately 1 ml of PBS was then added for microscopic visualization. Images were processed using a 4× objective lens, with transmitted bright-field light. For quantification, ORO stain particles were eluted with 100% isopropanol and analyzed using Thermo Fisher Scientific Varioskan Flash for spectrophotometry readings at 514 nm. Images were taken using light microscopy (×4 magnification, Leica).

The chorioallantoic membrane (CAM) of fertilized Japanese quail (Coturnix japonica) eggs (a breeding colony of IABG SAS) was used as a pre-clinical model system. The eggs were incubated in a forced draught incubator (MIDI BIOS, Sedlcany, Czech Republic) at 37.5 °C and 50–60% relative humidity. The ex ovo cultures were prepared as follows: The surface of the eggs was disinfected with a 70% ethanolic solution at embryonic day 3 (ED3) in a sterile laminar flow hood. Subsequently, the eggs were opened with scissors, and the embryos were transferred into the six-well tissue culture plates (Sarstedt, Germany) and kept in a 37 °C humidified incubator (Memmert, Buchenbach, Germany) until ED11.