Cell Hypoxia

To calculate single-sample gene set enrichment, we used the GSVA program to derive the absolute enrichment scores of gene sets from several publications and previously experimentally validated gene signatures from MsigDB [74 (link)] as follows: tumor proliferation signature [27 (link)], tumor inflammation signature [75 (link)], cellular response to hypoxia, MYC targets, DNA replication, G2M checkpoint, P53 pathway, PI3K/AKT/mTOR pathway, IFNG signaling, genes up-regulated by ROS, DNA repair, degradation of ECM, collagen formation, angiogenesis, EMT markers, apoptosis, TGFB signaling, hallmark glycolysis, hypoxia signature and metabolism related pathways. To derive the GSVA score of each signature in each tumor sample, the normalized log2 (TPM+1) values were passed on as input for GSVA in the RNA-seq mode. Differentially enriched gene sets between the glycolysis high and low tumor groups were defined by GSVA adj.p < 0.05 (we used the limma R pachage because the GSVA scores were normally distributed around zero) [76 (link)].

The HK-2 cells were plated in 35 mm dishes at a density of 10⁶ cells/ dish and incubated until they reached approximately 90% confluence for experiment. The cells in hypoxia/ reoxygenation (H/R) group were cultured for 12 h under hypoxic conditions (1% O₂, 94% N₂, and 5% CO₂) in medium without nutrients (glucose-free, serum-free) to induce hypoxic injury. After hypoxic treatment, the cells were transferred back to regular culture medium with oxygen for 2 h for reoxygenation. Control cells were incubated in complete culture medium in a regular incubator (5% CO₂ and 95% air). The cells in HCQ-treated groups were pretreated with HCQ (0.5, 2.5, and 5 μmol/L) for 12 h before H/R operation.

Hypoxia and reoxygenation were performed using OxyGenie mini-incubator (Baker, USA), which is based on our previous studies^{26 ,48 (link),49 (link)}. The portable cell culture incubator includes a battery-operated temperature controller with a heat plate, two prefilled and replaceable gas cylinders and a flow divider to hold six individual 1-well chamber assemblies on a glass plate (Fig. 6a). The 1-well assembly on an MEA plate fits to a MultiChannel Systems signal amplifier allowing long-term recordings outside an incubator. Portability of the OxyGenie and the individual 1-well chambers enable sample preparation and collection one at a time, minimizing the cell culture exposure to ambient air.Figure 6

(a) Portable cell culture instrument include battery operated temperature controller and heat plate, two gas cylinders and flow divider allowing six individual 1-well assemblies. 1-well assembly includes 1-well chamber on plate (glass or MEA), the lid and the lid lock to seal the cell culture and to avoid evaporation, the cover and the cover lock to create and maintain the gas environment around gas permeable 1-well chamber. 1-well assembly on MEA plate can be fitted also to MCS signal amplifier and thus use it for long-term recordings outside the incubator. (b) Hypoxia and hypoxia-reoxygenation protocol.

Day before exposing cells to hypoxia, serum- and glucose-free EB medium (glucose-free DMEM (Gibco) containing 1% MEM NEAA, 1% GlutaMAX and 0.5% Penicillin/streptomycin) was changed. On the following day, the samples were loaded into pre-warmed (37 °C) OxyGenie mini-incubator and hypoxia was initiated using 0% O₂ and 5% CO₂ (hypoxic) gas. The used time periods included 6, 8, 10, 12 and 24 h. Control samples were cultured in serum- and glucose-free EB medium in a standard 5% CO₂ incubator without the lids and covers for the same time periods.
Hypoxia-reoxygenation experiments were performed similarly to the hypoxia experiments, but after 8 or 24 h hypoxia, the gas was exchanged to 19% O₂ and 5% CO₂ (normoxic) gas for 24 h. The control cells were kept in the 1-well chambers on the glass inside the incubator for the whole experiment. Samples were collected after the hypoxia or hypoxia-reoxygenation one by one to minimize the exposure to ambient air. pH was measured from cell culture medium during sample collection using Sentron SI600 pH meter with Sentron MicroFET pH probe.
With MEA plates, hypoxia-reoxygenation was performed so that first the samples were connected to normoxic gas to measure a baseline overnight, after which, hypoxia was initiated using the hypoxic gas for 24 h. After the hypoxia, reoxygenation was performed by connecting the samples again to the normoxic gas for another 24 h. Hypoxia and hypoxia-reoxygenation protocols are presented in Fig. 6b.

The trophoblast cell line HTR8/SVneo were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China.) and cultured in DMEM-H medium (Gibco, USA) containing, 100 U/ml Penicillin–Streptomycin (Gibco, USA), and 10% fetal bovine serum (FBS, Gibco, USA). Cells which cultured at 37℃ and 5%CO2 conditions were adopted as normal control. And cells in the hypoxia group were placed under hypoxia (1% O2) in anoxic box, which can freely adjust oxygen concentration and flow meter detection, closed and cultured at 37℃ and 5%CO2. Cells were collected 48 h later for subsequent experiments.

Cell growth and viability were determined using a CCK-8 assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). SK-MEL-2, SK-MEL-28, and A2058 cells were seeded at 3 × 10³ cells per well in 96-well plates containing 130 μL medium. After overnight incubation, the cells were treated with 0.1% DMSO or PCI-34051 at various concentrations (0.1, 0.3, 1, 3, 10, 30, 100 µM) for 24 h, 48 h, and 72 h. The A2058 HDAC8 KO and overexpression (OE) cells were seeded at 3 × 10³ cells per well in 96-well plates containing 130 μL medium and incubated for 24 h, 48 h, and 72 h. Hypoxia was induced using 100 µM CoCl₂ or by placing the cells in a hypoxia induction chamber. Then, 13 μL of the CCK-8 reagent was added to each well for 3 h. Absorbance was measured at 450 nm using a multimode microplate reader (Tecan Group, Ltd., Mannedorf, Switzerland). The results were quantified relative to the control, and they are presented as percentages. The half-maximal cell viability inhibition concentration (IC₅₀) and half-maximal growth inhibition concentration (GI₅₀) were determined using GraphPad Prism ver. 7.0 software (GraphPad Software, San Diego, CA, USA).