Streptomycin

SH‐SY5Y cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% foetal bovine serum (FBS, Gibco), 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco). Primary cultured HBMECs were purchased from ScienCell Research Laboratories. HBMECs were cultured in Endothelial Cell Medium (ECM, ScienCell) supplemented with 5% FBS (ScienCell), 1% ECGS (ScienCell) and antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin, ScienCell). Cells were then incubated in a humidified atmosphere containing 5% CO2 at 37°C. NBP was diluted to a stock solution of 10 mmol/L in DMSO. Cells were treated with OGD. For OGD, normal growth medium was replaced with FBS‐free ECM, and cells were incubated in an anaerobic chamber for 6 hours in which the oxygen level remained below 0.5%. After OGD, cells continued to incubate for 12 hours under normal culture conditions. NBP (10 μmol/L) pre‐treatment lasted for 2 hours before OGD and continued during the re‐oxygenation process. To further estimate the effect of autophagy activation on OGD, cells were pre‐treated with RAPA (100 nmol/L) and 3‐MA (5 μmol/L) for 1 hour.

HUVECs (C-003-5C, American Type Culture Collection, Manassas, VA, USA) were grown in the endothelial cell medium (ECM) (ScienCell, Carlsbad, CA, USA) supplemented with 1% endothelial cell growth supplement (Sciencell) and 20% fetal bovine serum (ScienCell), 100 units/mL penicillin, and 100 μg/mL streptomycin (ScienCell). All cells were cultured in a humidified incubator with 5% CO₂ at 37 °C. For all experiments, HUVECs were used between the third and fifth passages and were starved in serum-free ECM for 4 h before drug treatment.

Human OSCC cell lines (SCC9 and Cal27) were obtained from the American Type Culture Collection (ATCC). HOK cells were obtained from ScienCell, and HN4 and HN6 cells were from patients with head and neck squamous cell carcinoma (HNSCC). HN4, HN6, SCC9 and Cal27 cells were cultured in Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12, Gibco, USA) containing 10% fetal calf serum (FBS, HyClone, USA), 100 U/ml penicillin and 100 μg/ml streptomycin (Invitrogen, USA). HOK cells were cultured in oral keratinocyte medium (OKM, ScienCell, USA) with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were incubated in a humidified atmosphere at 37 °C in the presence of 5% CO₂. All cell lines were passaged for fewer than 6 months.

The tube-like structures of HUVECs were developed on growth factor-reduced Matrigel (BD Bioscience, USA) in conditioned media and were assayed using Transwell plates with polycarbonate filters (pore size: 4 μm). Before the experiment, the Matrigel sterilized tips were chilled at 4 °C overnight. Twenty-four-well Transwell cultivation plates were used and daubed with the suspension of 200 μL Matrigel and 200 μL complete medium according to the manufacturer’s instructions. HUVECs were seeded in endothelial conditioned medium containing 10% FBS, 2 mM/L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/mL streptomycin, and 1% ECGS (ScienCell, CA, USA) for 12 h and plated onto the lower layer of the Transwell with diluted Matrigel at a density of 2 × 10⁵ cells/mL/well. Then the cells treated as per the experimental design were loaded into each of the upper wells. The Matrigel in the Transwell cultivation was incubated at 37 °C and 5% CO₂ for 6 h. HUVECs were stained using 2 μM calcein AM fluorescent dye (Solarbio, Beijing, China) (Fig. 6c). Tube areas were quantified by the number of tubes and relative areas of tubes. The results were recorded under the microscope at 6 h. The number of tubes and relative area of tubes were assessed from five figures of each well by Adobe Photoshop (Adobe, San Jose, CA, USA).

Human renal mesangial cells were purchased from ScienCell™. These cells were cultured as reported previously.²² Briefly, cells were plated on poly‐l‐lysine‐coated culture vessels with mesangial cell medium (MsCM) (ScienCell™) supplemented with 2% FBS (ScienCell™), 100 U/mL penicillin, 100 μg/mL streptomycin (ScienCell™) and 1% mesangial cell growth factors (ScienCell™). Following starvation in serum‐free MsCM for 24 hours, cells were washed and stimulated with 0.1 ng/mL human IL‐6 (200‐06; Peprotech) for 12, 24 or 36 hours.²³ Control cultures were incubated with MsCM only.