Sh sy5y neuroblastoma cells

The D4MB-6 viral variant was obtained following a published protocol [9 (link)]. Briefly, Aedes albopictus C6/36HT cells were inoculated with a DENV-4 serotype isolate, and the supernatants were harvested. This virus was inoculated three successive times into SH-SY5Y neuroblastoma cells (ATCC). The third passage was intracerebrally inoculated into Balb/C suckling mice, recovered six days later, and then re-inoculated five more times to obtain the dengue serotype 4 mouse brain passaged six times strain (neuro-adapted D4MB-6). Brains were dissected from anesthetized mice, homogenized in 10% tissue suspensions and titrated using a plaque assay into LLMCK2 (ATCC) cells (2.6 X 10⁶ PFU/ml). Non-infected brain lysate or C6/36HT supernatants were used as mock inocula.

SH-SY5y neuroblastoma cells (ATCC CRL-2266, Manassas, VA, USA) were purchased from the Korean Cell Line Bank (Seoul, Korea). The cells were identified with STR profiling, including D5S818, D13S317, D7S820, vWA, FGA, TH01, TPOX by Korean Cell Line Bank before distribution. The cells were cultured in DMEM medium (Welgene, Gyeongsan-si, Korea) supplemented with 10% fetal bovine serum (FBS), penicillin G (100 units/mL), streptomycin (100 μg/mL), and l-glutamine (2 mM), and grown at 37 °C in a humidified incubator containing 5% CO₂ and 95% air. Cells were maintained within passage #15 prior to any experiments.

Human SH-SY5Y neuroblastoma cells (A.T.C.C., Manassas, VA, USA) were cultured in DMEM, supplemented with 10% FBS, 1.0 mM glutamine and antibiotics. Cell cultures were maintained in a 5.0% CO₂ humidified atmosphere at 37 °C and grown until they reached 80% confluence for a maximum of 20 passages.

SH-SY5Y neuroblastoma cells (purchased from ATCC CRL-2266) were plated at 3,000 cells/mL and grown in T182 tissue culture flasks (adherent and floating cells were observed). After 72 h, cells were infected with 100 MOI of EV-D68 strain US/MO/14-18947 at 33°C. After 1 h of incubation with virus, supernatant was removed (along with floating cells), adherent cells rinsed with PBS, and 40 mL of media (95% high-glucose DMEM + 5% fetal bovine serum) was added onto cells for 12 days at 33°C. At 3-day intervals, 10 mL of medium was removed from flask and frozen prior to density centrifugation and analysis.

SH-SY5Y neuroblastoma cells were obtained from ATCC (Manassas, VA). Human telomerase-immortalized retinal pigmented epithelial (RPE) cells were kindly provided by Dr. Jun Kim (KAIST, South Korea). Wild-type (WT) MEFs, as well as Drp1 and OPA1 knockout MEFs were generously provided by Dr. Katsuyoshi Mihara (Kyushu University, Japan) and Dr. Joo-Yong Lee (Chungnam National University, Korea). MEFs including a doxycycline-induced deletion of ATG5 (M5-7 cells) were kindly provided by Dr. Noboru Mizushima (Tokyo University, Japan), with ATG5 depletion being induced by maintaining the cells in doxycycline (1 μg/ml) containing medium, for 4 days. AMPK double knockout (AMPK DKO) MEFs were kindly provided by Dr. Benoit Viollet (Université Paris Descartes, France) and maintained in doxycycline-containing culture medium. To generate stable cell lines, SH-SY5Y cells were transfected with pEGFP-LC3 (SY5Y/GFP-LC3 cells), pEGFP-TFEB (SY5Y/GFP-TFEB cells), and pMito-HyPer (SY5Y/Mito-HyPer) using Lipofectamine 2000 in accordance with the manufacturer’s protocol (#11668019, Thermo Fisher Scientific, Waltham, MA). Transfectants were selected by growth in medium containing 1 mg/ml of G418 (#10131027, Thermo Fisher Scientific) for 7 days. After single cell dropping, the stable clones were selected under a fluorescence microscope (IX71, Olympus, Tokyo, Japan).

Human SH-SY5Y neuroblastoma cells (A.T.C.C.,
Manassas, VA) were cultured in Dulbecco’s modified Eagle’s
medium (DMEM)-F12+GlutaMax supplement (Thermo Fisher Scientific, Waltham,
MA) with 10% heat-inactivated fetal bovine serum. The cell cultures
were maintained in a 5.0% CO₂ humidified atmosphere at
37 °C and grown until 80% confluence for a maximum of 20 passages.