Sk n sh cells

Porcine kidney stable (PS) cells [30 (link)] were cultured at 37 °C in Leibovitz (L-15) medium with L-glutamine, supplemented with 3% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich, Czech Republic). Human neuroblastoma SK-N-SH cells (obtained from ATCC) were cultured at 37 °C in 5% CO₂ in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 292 μg/mL L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich, Czech Republic).
Human primary astrocytes (HBCA cells; purchased from ScienCell at passage 1) were grown following the supplier’s instructions in astrocyte medium with astrocyte growth supplement, 6% fetal bovine serum and 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich, Czech Republic). The cells exhibited typical astrocyte morphology and expressed GFAP, as confirmed by staining with GFAP antibody conjugated to Alexa Fluor 488 (1 : 100; Santa Cruz).

Referring to previously published literature [21 (link)], SK-N-SH cells (ATCC, Manassas, VA, USA) were placed in Dulbecco’s modified Eagle medium (DMEM) (Invitrogen, Carlsbad, CA, USA) (containing 10% fetal bovine serum and 100 U/mL penicillin/streptomycin) with humidified air of 37°C and 5% CO₂ for 48 h. Next, the SK-N-SH cells were seeded into 6-well plates at a density of 6 × 10⁴ cells/cm², followed by transfection with 2 μg pCDNA3.1-SNHG16 (Genecopoeia, Guangzhou, China), 100 nM miR-421 mimic (Thermo Fisher Scientific, Waltham, MA, USA), 50 nM si-XIAP-1 and si-XIAP-2 (Shanghai BGI Technology Co., LTD, Shanghai, China) and equal volumes of corresponding negative controls using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). Prior to OGD/R treatment, the cells were cultured for 48 consecutive h, and the transfection efficiency was analyzed by means of reverse transcription quantitative polymerase chain reaction (qRT-PCR). Subsequently, the transfected cells were deprived of the normal medium, rinsed with phosphate buffer saline (PBS) 3 times, and cultured in serum/glucose-free DMEM. Afterward, the cells were placed in a hypoxic chamber (containing 95% N₂ and 5% CO₂) and then cultured under normal conditions for 12 h to induce OGD/R injury. Simultaneously, control cells were cultured at 37°C with 5% CO₂ in air without any special treatment.

SK-N-SH cells (ATCC, Manassas, VA) were cultured and grown in Dulbecco's Modified Eagle's Medium (DMEM; Sigma-Aldrich, St. Louis, MD) supplemented with 10% Fetal Bovine Serum (FBS; Invitrogen, Carlsbad, CA). SH-SY5Y cells (ATCC) were maintained in 1:1 mixture of Hams F12 medium (Sigma-Aldrich) and Minimum Essential Medium (MEM, Sigma-Aldrich) supplemented with 15% FBS, 2 mM L-glutamine (Life Technologies, Tokyo, Japan) and 1% non-essential amino acids (NEAA, Sigma-Aldrich). Cells were grown at 37°C and under 5% CO₂. Cells from the 3rd passage were used for drug experiments.

Vero cell line (African green monkey kidney epithelial cells, ATCC CCL-81), HeLa cell line (ATCC, CCL-2), HEp-2 cell line (ATCC, CCL-23) and human neuroblastoma SK-N-SH cells (ATCC, HTB-11) were purchased from ATCC (Manassas, VA) or from Cell Bank of Chinese Academy of Sciences (Shanghai, China). Mouse embryo fibroblast (MEF) cells (isolated from C57BL/6J mouse embryos) were prepared in the lab following published protocol [64 (link)]. The cells were cultured in DMEM (high glucose) supplemented with 10% heat-inactivated fetal bovine serum (FBS), sodium pyruvate and non-essential amino acids (Life Technologies, Carlsbad, CA) in a humidified incubator at 37°C with 5% CO₂. HSV-1 strain F and HSV-2 strain G were propagated and titrated on Vero cells.

Vero cells were purchased from the American Type Culture Collection (ATCC), and were cultured in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS) (GBICO) and antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin). SK-N-SH cells were also purchased from the ATCC, and were cultured in Dulbecco’s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F-12) supplemented with 10% FBS and antibiotics (100 U/ml penicillin G and 100 mg/ml streptomycin).
EV71 strain SHZH98 isolated from the throat swab sample of an HFMD case occurring in 1998 in China was kindly provided by Dr. Qi Jin, Institute of Pathogen Biology, Chinese Academy of Medical Science and Peking Union Medical School, Beijing, China. EV71 strain BrCr (VR-1775) and H (VR-1432) were purchased from the ATCC. EV71 strain JS-52 was a kind gift from Dr. Xiangzhong Ye, Beijing Wantai Biological Pharmacy Enterprise Co., Ltd. EV71 strain SHZH98 was passaged in Vero and SK-N-SH cells. EV71 strains BrCr, H, and JS-52 were passaged in Vero cells. CVB2 (strain Ohio-1), CVB3 (strain Nancy) and CVB6 (strain Schmitt) were all got from the ATCC and passaged in Vero cells.

SK-N-SH cells and HEK293 cells were purchased from ATCC. HEK293/APPswe cells were transfected, selected with antibiotics (G418, 1 mg/ml), and maintained in lab. All of these cell lines were cultured in Modified Eagle’s Medium (MEM) with 10% (w/v) heat-inactivated foetal bovine serum (FBS) in a humidified incubator with 5% CO2/95% air (v/v) at 37 °C. Human iPSC-derived NSCs were provided by IxCell Biotechnology., Ltd. and maintained with neural stem cell culturing basal medium containing 1:50 NSC supplement (I × Cell).
The human HA-tagged HTR6 construct was kindly provided by Dr. Xie Xin (SIMM, China). The viral constructs encoding the HT6R or F20 were generated by subcloning the cDNA into a FUGW vector using the BamHI restriction sites. The constructs were verified by sequencing. The primers used are listed in Supplementary Table 1.
For RNA interference (RNAi) experiments, shRNA targeting human HTR6, HTR2B, HTR4, HTR7, DRD2, Gα_s, ARRB2 or CDK5 was cloned into a pLKO.1 vector following the online protocol (Addgene, http://www.addgene.org/tools/protocols/plko/). All targeting sequences are listed in Suppplementary Table 2. A pLKO.1-sh-SCRAM vector expressing a scrambled sequence complementary to no human gene was used as a control.