H35 hypoxystation

To determine protein expression after hypoxia, cells were incubated under hypoxic conditions (0.5% O₂, H35 hypoxystation, Don Whitley Scientific Ltd., West Yorkshire, UK) for 1 h, 4 h, 8 h, 16 h or 24 h. To determine protein expression after AKT inhibition, cells were treated overnight (16 h) with 0 or 2 μM MK-2206 (Selleckchem, Houston, TX, USA) under standard normoxic conditions and thereafter incubated under normoxic conditions or under hypoxic conditions (0.5% O₂, H35 hypoxystation, Don Whitley Scientific Ltd., West Yorkshire, UK) for 4 h, 8 h or 16 h.
To assess cell survival after hypoxia and/or AKT-inhibition, cells were seeded in 96-well plates. After the cells were allowed to attach overnight under standard normoxic conditions, 0, 0.5, 1, 2, 4 or 8 μM, MK-2206 was added and cells were incubated under normoxic conditions or under hypoxic conditions (0.5% O₂) for 72 h. To prevent interference of hypoxia in the determination of cell survival with the cell-counting Kit-8 assay (Sigma–Aldrich Chemie BV, Zwijndrecht, The Netherlands), cells were placed back into normoxic conditions before measurement of cell survival. Cell survival was measured 72 h after hypoxic incubation in HNSCC lines and 24 h after hypoxic incubation in NSCLC lines.

To induce simulated cold ischemia-reperfusion injury, an in vitro hypothermal H/R model was used. Cell culture medium of plated H9c2 cells was aspirated and cells were washed once using PBS. PBS was then added to each well (1 mL/well for 6-well plates; 100 μL/well for 96-well microplates) before plates were placed in the HypOxystation H35 (Don Whitley Scientific, Shipley, UK) set at 10 °C and chamber conditions of 0.5% O₂, 4.0% CO₂, and 95.5% N₂ for 18 h. Cells were then removed from the HypOxystation H35 and complete DMEM medium with 10% FBS was added to each well (1 mL/well for 6-well plates; 100 μL/well for 96-well microplates). Cells were then incubated for 24 h at 37 °C and 5% CO₂. Cells in the normoxia group were incubated at 37 °C and 5% CO₂.

Human melanoma cell lines MelAKR and MelJUSO and the murine melanoma cell line B16.F10 were cultured in RPMI 1640 (Gibco). The human melanoma cell lines MelWBO and Mel136.2 were cultured in IMDM (Gibco). The human melanoma cell line Mel88.23 and human cervical cancer cell line HeLa were cultured in DMEM (Gibco). All media were supplemented with 8% heat-inactivated fetal calf serum (FCS; #0270–106), 1% penicillin/streptomycin (P/S, #15140122) and 2 mM L-glutamine (#25030024, Thermofisher Scientific). All cell lines have been described before [21 (link), 23 (link), 24 (link)] and were cultured at 37 °C and 5% CO₂, and at atmospheric oxygen levels (normoxia). For hypoxia, cells were incubated in the H35 Hypoxystation (Don Whitley Scientific) at 1% O₂. One day prior to stimulation, cells were seeded in a total volume of 2 ml per well using 6-well plates (Greiner Bio One, #657160) at a cell density Mel88.23 (2.5 × 10⁵/well), Mel136.2, MelWBO and B16.F10 at 1 × 10⁵/well, MelAKR, MelJUSO, HeLa at 1.5 × 10⁵/well. Cells were exposed to hypoxia, incubated with IFNγ (Roche, #11040596001) or HIF-stabilizing agent DFO (Deferoxamine mesylate salt, Sigma-Aldrich, D9533), or a combination thereof, as indicated in the results. DFO-containing medium was replaced after 24 h.