Streptomycin

A three-layer, three-dimensional co-culture system was created by connecting a six-well Transwell cell culture plate to a 12-well Transwell membrane. First, 2 × 10⁵ human cerebral microvascular endothelial cells (HCMEC/D3, Jennio Biotech Co., Ltd., Guangzhou, China) were seeded in the upper 12-well Transwell membrane (upper chamber) and cultured in a routine incubator at 37 °C for two days until tight junctions between the cells were formed, to obtain a blood-brain barrier. On the second day, 1.2 × 10⁵ astrocytes (SVG P12, BLUEFBIO^TM, Shanghai, China) and 4.5 × 10⁴ human microglia (HMC3, BLUEFBIO^TM, Shanghai, China) were seeded in the medium layer of a six-well Transwell membrane (medium chamber), and 2 × 10⁵ human hippocampal neurons (HPPNCS, BLUEFBIO^TM, Shanghai, China) were seeded in the bottom layer (lower chamber). All four cell types were inoculated into high-glucose Dulbecco’s Modified Eagle Medium (DMEM), containing 10% fetal bovine serum (Procell Life Science&Technology Co., Ltd., China) with 1% penicillin and streptomycin (Procell Life Science&Technology Co., Ltd., China, China), for 20–24 h to achieve normal cell morphology. The three-dimensional multicell co-culture model was subjected to OGD on the third day.

HCECs (Purchased from BNCC, Beijing, China) were cultured in MEM medium (Basal Media, Shanghai, China) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and Penicillin–Streptomycin Solution (10 kU/ml Penicillin, 10 mg/ml Streptomycin, Procell, Wuhan, China) in a humidified incubator at 37 °C containing 5% CO₂. According to the groups, D-Glucose (Procell, Wuhan, China) was dilated to different concentration. The whole research procedure was approved by the Ethics Committee of Chongqing Medical University and was performed following the Declaration of Helsinki.

HEK293 cells were cultured at 37 °C in a humidified incubator with 5% CO₂ in a high-sugar medium (Dulbecco’s Modified Eagle medium (DMEM), HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS, Hyclone, Waltham, MA, USA), 100 U/mL penicillin, and 100 μg/mL streptomycin (Procell, Wuhan, China). AjGPER1-EGFP, CALR-RFP, or TGN38-RFP vectors were transfected alone or cotransfected into HEK293 cells using X-tremeGENE HP DNA transfection reagent (Roche Applied Science, Indianapolis, IN, USA) as required. The cells were then incubated in 6-well plates for 24 h and inoculated into 24-well plates for subsequent experiments. To eliminate the effects of FBS, HEK293 cells were starved in FBS-free DMEM for 4 h prior to experiments.

Human PCa cell lines (PC3 and C4-2) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) with 10% fetal bovine serum (FBS, Cat# 10270-106, Gibco, UK) and penicillin–streptomycin combination solution (10 kU/mL penicillin and 10 mg/mL streptomycin, Cat# PB180120, Procell, Wuhan, China) at 37 °C and 5% CO₂. PCa cells were transfected with small interfering RNAs (siRNAs) (General biological system, Anhui, China) using Lipofectamine 3000 reagent (Lot# 2307436, Invitrogen, USA). The sequences of si-cyclin-dependent kinase inhibitor 2C (si-CDKN2C), si-Rac GTPase-activating protein 1 (si-RACGAP1), and the nontargeting control (NC) are shown in Additional file 1: Table S1. The antibodies used were as follows: anti-CDKN2C (Cat# ab192239, Abcam, Cambridge, UK), anti-RACGAP1 (Cat# ab134972, Abcam, Cambridge, UK), anti-β‐actin (Cat# AF7018; Affinity, OH, USA), and anti-β‐tubulin (Cat# AF7011, Affinity, OH, USA). The secondary antibodies included goat anti‐rabbit for CDKN2C, RACGAP1, and β-actin (Cat# AS014, ABclonal, Wuhan, China) and goat anti-mouse for β-tubulin (Cat# S0002, Affinity, OH, USA).