Mdivi

Cells (10⁵/mL) were cultured to monolayer confluence in 60-mm culture dishes for 20 h, rinsed twice with PBS, then transfected with either pFlag or pFlag-B2 for 2 h. Cells were then treated with either 1 mM NAC or 5 µM Mdivi (both Sigma) at 28 °C for 48 and 72 h. After the medium was removed, the monolayers were washed with PBS, incubated with 100 μL of PI (Boehringer-Mannheim Mannheim, Germany) in HEPES buffer for 10–15 min and evaluated using fluorescence microscopy as previously described [26 (link)].

The detailed induction protocol has been described previously [10 ]. In brief, the induction solution consisted of 0.3× Danieau’s solution with 5 µM 4-OHT and 0.5% DMSO to enhance penetration of 4-OHT. A 10 mM stock of 4-OHT dissolved in 96% ethanol was stored at −20 °C protected from light. At 48 h post-fertilisation (hpf), selection marker positive (green heart) larvae were transferred to petri dishes containing 20 ml 4-OHT induction solution, at 50 larvae per dish. The larvae were then maintained at 28.5 °C in the dark. For Oxygen consumption rate (OCR) and glycolysis analysis using Seahorse ® analyser, the larvae were anaesthetised with 55 mg/L eugenol (Sigma) at 22 h post-induction (hpi), and screened. From the same clutch, larvae with GFP positive skin cells were collected as pre-neoplastic cell (PNC) group, and larvae with GFP negative skin were collected as Wild Type (WT) siblings. For drug treatment, larvae were screened at 8hpi, and positive larvae from CAAX group were used as controls. The sorted embryos carrying PNCs were randomly placed in fresh 4-OHT induction solution with appropriate drugs or vehicle control. IACS-010759 (Selleckchem, Catalog No.S8731) were used at 1 uM, metformin (Sigma-Aldrich, Product number: 31724) 50 uM, mdivi 1 µM (Sigma-Aldrich, product number: 475856) in induction solution.