D glucose

To investigate gene and protien expression changes compared with the general culturing conditions of primary cortex cultures, glucose starvation and derpivation were performed, as well as glucose starvation followed by refeeding. Glucose starvation of the cell cultures was performed by changing media to NeurobasalA with 0 g/L D-Glucose (A2477501, Thermo Fisher Scientific), 2 mM GlutaMax, HEPES, 1% Pen-strep and 1x B27 without insulin (A1895601, Thermo Fisher Scientific). Starvation was performed for 3 and 12 h. The starved and refed group was subjected to the same condition as the starved group for 3 and 12 h, before refeeding for 12 h on regular NeurobasalA media containing 4.5 g/L D-Glucose, 2 mM GlutaMax, Hepes, 1% Pen-strep and 1x B27. The control groups were kept on NeurobasalA media, but underwent equal number of media change as the corresponding experimental group.
Sugar deprivation of the cell culture was performed by changing the media to DMEM containing 1 g/L D-Glucose (11054020, Thermo Fisher Scientific), 2 mM GlutaMax, HEPES, 1% Pen-strep, and 1x B27. The deprivation was performed for 3 and 12 h. The control group was kept on DMEM containing 4.5 g/L D-Glucose (same concentration as in NeurobasalA Media), 2 mM GlutaMax, HEPES, 1% Pen-strep and 1x B27 Supplement (all from Invitrogen) and underwent equal number of media changes as the deprived cells.

For the study of glutamine deficiency, special DMEM medium from GIBCO, United States, which lacks D-glucose, L-glutamine, sodium pyruvate, and phenol red, was used, and also lacks L-asparagine. The concentrations of all amino acids in the DMEM medium used are detailed in Table 1. The medium was supplemented with 1 g/L D-glucose (Sigma, United States) and 2% dialyzed fetal bovine serum (Hyclone, United States). For glutamine depletion rescue experiments, 1 g/L D-glucose, 2 mM L-glutamine (GIBCO, United States), and 2 mM L-asparagine were added to the medium when necessary. All cells were incubated at 28°C in an incubator with 5% CO₂. The cells were washed with 1 × phosphate-buffered saline (PBS, VWR) before exposure.

African green monkey kidney cells (Vero E6; ATCC CRL-1586) and African green monkey kidney cells expressing human Transmembrane Serine Protease 2 and human Angiotensin-Converting Enzyme 2 (Vero E6-TMPRSS2-T2A-ACE2 cells; NR-54970) were maintained in DMEM with 4.5 g/L of D-Glucose (Dulbecco’s Modified Eagle’s Medium—Gibco^TM, Billings, MT, USA), 1 mM sodium pyruvate, supplemented with 10% fetal bovine serum (ThermoFisher Scientific, Waltham, MA, USA), and 0.75% w/v sodium bicarbonate. Human lung adenocarcinoma epithelial cells (Calu-3, ATCC HTB-55) were maintained in DMEM with 1.0 g/L of D-Glucose (Dulbecco’s Modified Eagle’s Medium—Gibco^TM, Billings, MT, USA), 1mM sodium pyruvate, supplemented with 10% fetal bovine serum (ThermoFisher Scientific, Waltham, MA, USA), and 0.75% w/v sodium bicarbonate. Cells were cultured at 37 °C with 5% CO₂.

The cell model with diabetes was induced by HG as previously described by Qiu^{22 (link), 23 (link)}. Briefly, BSMCs were grown in DMEM supplemented with 5 mM D-glucose (normal glucose as a control), to mimic diabetes in vitro, the cells were maintained in DMEM with 25 mM D-glucose (Gibco/BRL) for 48 h as a HG treatment. BSMCs were divided into 8 groups: control group, HG group, HG + CGRP group, HG + CGRP + AA group, CGRP group, AA group, HG + CGRP + CGRP-8-37 group and HG + LY2228820 group. The relevant use concentrations were as follows: CGRP (20 pg/ml, Abcam, UK), AA (5 μM, Abcam, UK), CGRP-8-37 (2 μM, Abcam, UK) and LY2228820 (1 μM, Abcam, UK). The cells were incubated in the incubator with 5% CO₂ at 37 °C for 48 h.

HeLa cells (passage 20) and human dermal fibroblasts (HDF, passage 7) were cultured in 75 cm² flasks (Greiner Bio One) and maintained under standard cell culture conditions at 37 °C and with a 5% CO₂ humidified atmosphere prior to experiments. Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 g/L of D-Glucose (Gibco, Life Technologies, UK) was used for HeLa cells and Dulbecco’s Modified Eagle’s Medium (DMEM) with 1 g/L of D-Glucose used for HDF cells, both media supplemented with 10% FBS (Gibco, Life Technologies, UK) and 1% penicillin/streptomycin (Gibco, Life Technologies, UK) under sterile conditions.

Pooled Human Umbilical Vein Endothelial Cells (HUVECs) (Lonza Inc, USA or Promocell, UK), grown in endothelial basal cell medium 2 supplemented with EGM-2 Singlequot supplements (Lonza Inc, USA). HUVECs maintained at 37 °C in a humidified atmosphere of 5% CO₂ and used between passages 1–5. Human Embryonic Kidney 293 cells (HEK293T—Invitrogen, UK) were grown in Dulbecco's modified Eagle medium (DMEM—Invitrogen, Paisley, UK) containing D-glucose, L-glutamine, and pyruvate (Life Technologies, UK) supplemented with 10% Fetal Bovine Serum (FBS—Sigma‐Aldrich), 100 U/m penicillin, and 100 mg/ml streptomycin (Sigma‐Aldrich). Cos-7 cells were obtained from the American Type Culture Collection (ATCC) and maintained in DMEM supplemented with 10% FBS and 100 U/ml penicillin + 100 mg/ml streptomycin. Cells were maintained at 37 °C and 5% CO₂ in a humidified incubator and used between passage 2 and 20.