Pt2385

Hydroxychloroquine, temsirolimus (CCI-779), and 5-fluorouracil (5-FU) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The HIF-2α-specific inhibitor PT-2385 was purchased from Biovision. Dihydroethidium (DHE) was obtained from Invitrogen^TM (Waltham, MA, USA) (Cat. No. D11347). N-Acetyl-L-cystein (NAC) was obtained from Sigma-Aldrich (Cat. No. A7250). The antibodies used in the experiments were from the following sources: anti-LC3 from Novus Biologicals (Centennial, CO, USA); and anti-activated caspase-3, anti-HIF-1α, anti-HIF-2α, anti-SQSTM1/p62, anti-β-tubulin, anti-Atg7, anti-Phospho-S6 Ribosomal protein (Ser235/236), anti-Beclin.1 and anti-PI3K-Class III were all obtained from Cell Signaling Technology (Danvers, MA, USA). Goat anti-mouse and anti-rabbit IgG-horseradish peroxidase-conjugates were from Pierce (Rockford, IL, USA)

1.2 × 10⁴ 786–0 cells or 2 × 10⁴ RCC4 cells were seeded per well in 96-well ImageLock™ plates (4379, Essen BioScience) and incubated for 24 h. When the effect of PT2385 (B1920, BioVision) was tested, the compound was added to the wells once the cells were attached. After wounding, cells were washed with fresh media and plates were placed into the IncuCyte ZOOM® until wound closure. Scanning was performed using a 10x objective and scheduled every 2 h. Migration ability of the cells was analysed through two integrated metrics that the IncuCyte™ Software calculates based on the processed images: wound width and wound confluence. Wound width represents the average distance (μm) between the leading edge of the population of migrating cells (scratch wound mask) within an image. Wound confluence determines the percentage of wound area that is occupied by cells, and it relies on the initial scratch wound mask to differentiate the wounded from the non-wounded region.

PT2385 (Abcam) was initially dissolved in DMSO and then diluted 1:1000 in buffer with 20 mM Tris pH 8.0 and 150 mM NaCl. Temperatures were measured on a Nano ITC system (TA Instruments), with HIF-2α PAS-B domains in 20 mM Tris pH 8.0, 150 mM NaCl, and 0.1% DMSO at 20°C. 30 aliquots of 7.98 μL of 200 μM HIF-2α PAS-B domain, or the S305M mutation, were injected into 40 μM PT2385. Stoichiometry, binding constant, and change in enthalpy of interaction were calculated using the ITCRun and NanoAnalyze software (TA Instruments).