Penicillin streptomycin solution

Mouse neuronal N1E-115 and green monkey kidney epithelial COS-7 cells (JCRB Cell Bank, Osaka, Japan, and Japan Health Sciences Foundation, Osaka, Japan) were cultured on 6- or 10-cm cell culture dishes (ThermoFisher Scientific, Waltham, MA, USA) in high-glucose Dulbecco’s modified Eagle medium (DMEM; Nacalai Tesque) containing 10% heat-inactivated fetal bovine serum (FBS; ThermoFisher Scientific) and penicillin-streptomycin solution (Nacalai Tesque) in 5% CO₂ at 37 °C.
COS-7 and N1E-115 cells are high transfection efficiency and neuronal differentiation abilities, respectively [13 (link),14 (link)]. Cell lines stably expressing the wild-type (indicated as WT in the figures) Chmp2b gene or the gene with the T104N mutation were selected in the presence of G418 (Nacalai Tesque) as described previously [15 (link)] and isolated as a single clone. To induce differentiation, N1E-115 cells were cultured in DMEM and 1% FBS containing penicillin-streptomycin in 5% CO₂ at 37 °C for 48 h. Cells with processes longer than their cell body length were considered to be neurite-bearing cells (i.e., differentiated cells) [16 (link)]. Under these conditions, in each experiment, it was estimated that less than 5% of adherent cells incorporated trypan blue (Nacalai Tesque).

Dulbecco’s Modified Essential Medium/Ham’s Nutrient Mixture (DMEM:F12), Penicillin-Streptomycin solution and 2.5g/l-Trypsin/1mmol/l-EDTA Solution were purchased from Nacalai Tesque (Tokyo, Japan). Fetal bovine serum (FBS) and non-essential amino acids (NEAA) was purchased from Gibco-BRL (Grand Island, NY). Lipopolysaccharide (LPS) from Escherichia coli O55:B5 was purchased from Merck (Darmstadt, Germany).
SH-SY5Y neuroblastoma cell line were purchased from ATCC (ATCC^® CRL-2266^™). The cells were initially grown in Dulbecco’s Modified Essential Medium (DMEM:F12) which contains 4.5 g/l glucose with 2mM of L-glutamine and sodium pyruvate, supplemented with 15% FBS, 1% of Penicillin-Streptomycin mixed solution and 1% NEAA at 37°C with 5% carbon dioxide (CO₂). Then, the cells were induced with 10 μM of all-trans retinoic acid for 5 days in a differentiation media (DMEM:F12, supplemented with 2.5% Fetal bovine serum (FBS) and 1% of Penicillin-Streptomycin mixed solution) (Forster et al., 2016 (link); Izham et al., 2018 (link)).

Citric acid-1-hydrate (Bendosen, Laboratory Chemicals, Johor Bahru, Malaysia); ethylenediamine; glycerol; ethanol, 98%; hydrochloric acid, HCl 37%; phosphate-buffered saline (PBS) (QREC, Grade AR, (Asia) Sdn. Bhd, Rawang, Selangor, Malaysia); titanium(IV) tetraisopropoxide, TTIP ≥ 97% purity; commercial pure anatase, ≥99%; sodium pyruvate; dimethyl sulfoxide, DMSO; 1,4-benzoquinone (Sigma-Aldrich, Co., St. Louis, MO, USA); phosphate buffered saline (PBS) (Sigma-Aldrich, Co., Taufkirchen, Germany); Dulbecco’s modified Eagle’s medium, DMEM; penicillin-streptomycin solution, 10,000 units/mL (Nacalai Tesque, Nakagyo-ku, Kyoto, Japan); fetal bovine serum (FBS) (TICO Europe, DJ, Amstelveen, The Netherlands); CellTiter 96^® AQueous One Solution Cell Proliferation Assay (MTS); Caspase-Glo^® 3/7 Assay (Promega, Madison, WI, USA); 2′,7′-dichlorofluorescein diacetate (DCFDA, Merck-Millipore, Burlington, MA, USA); tetramethylrhodamine ethyl ester; TMRE-Mitochondrial Membrane Potential Assay Kit (Abcam, Trumpington, Cambridge CB2 0AX, UK) were used as purchased without any further purification.

Eight-week-old healthy male Sprague–Dawley rats were used to isolate TSPCs. All animal experimental protocols were approved by the Animal Research Committee of Tohoku University (approval number: 2020DnA-009). The TSPCs were isolated according to previously established procedures [36 (link)]. Briefly, the midsubstance of Achilles tendon tissues was collected. The collected tissues were minced and digested for 2.5 h at 37 °C with 3 mg/mL of type I collagenase (Sigma-Aldrich, St. Louis, MO, USA). Afterward, the cells were passed through a cell strainer (70 μm pore size, Becton, Franklin Lakes, NJ, USA) to obtain a single-cell suspension. The isolated cells were washed and centrifuged to resuspend in low-glucose Dulbecco’s Modified Eagle’s medium (FUJIFILM Wako Pure Chemical Co., Osaka, Japan) containing 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA) and 1% penicillin/streptomycin solution (Nacalai Tesque Inc., Kyoto, Japan) as the complete medium at 37 °C in a 5% CO₂ incubator. The cells at passages 3 and 5 (P3, P5) were used for all subsequent experiments. P5 cells served as a control group, and the stemness of TSPCs was identified by measuring the mRNA expression of CD44, CD90, and Nanog using a qPCR. The culture medium was replaced every 3 days during the experiments.

UM47 and UM104, the HPV16-positive head and neck carcinoma cell lines, were purchased from The University of Michigan. Caski was the gift from Department of Obstetrics and Gynecology, Osaka University Graduate School of Medicine. UM47 and UM104 were derived from lateral tongue carcinoma and oral cavity carcinoma respectively. These cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque), supplemented with 10% foetal bovine serum (FBS, Sigma), 1% non-essential amino acids (NEAA, Nacalai Tesque), 1% L-glutamine (Nacalai Tesque), and 1% penicillin/streptomycin solution (Nacalai Tesque). The HPV16-positive cervical carcinoma cell line, Caski, was cultured in DMEM, supplemented with 10% FBS and 1% penicillin/streptomycin. The human embryonic kidney cells, 293FT, were cultured in DMEM, supplemented with 10% FBS, 1% NEAA, 1% L-glutamine, and 1% sodium pyruvate (Sigma). These cells were incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO₂.

Dulbecco’s modified Eagle medium (DMEM), Ham's F12 Medium, Neurobasal A medium, B27 supplement, GlutaMAX, fetal bovine serum, Alexa Fluor secondary antibodies, LysoTracker, MitoTracker, Alexa Fluor 633-conjugated WGA, CellMask, and Hoechst33342 were obtained from Life Technologies (Carlsbad, CA). The penicillin/streptomycin solution and polyethyleneimine were from Nakalai Tesque (Kyoto, Japan). The HaloTag vectors and HaloTag ligands were from Promega (Madison, WI). The anti-KDEL antibody was from Enzo Life Sciences (Farmingdale, NY). The anti-Rab5 and anti-Rab7 antibodies were from Cell Signaling Technology (Danvers, MA). The G Silencer siRNA against rat GPR3 (5’-CCUACUACUGAGAGACAACtt-3’/5’-GUUGUGUCUGAGUAGUAGGtg-3’) and the negative control #1 siRNA were purchased from Ambion, Inc. (Austin, TX). The glass-bottomed culture dishes (35-mm diameter) were from MatTek (Ashland, MA) and the 8-well chamber slides were from AGC Techno Glass (Shizuoka, Japan). All other reagents or chemicals were from Sigma-Aldrich (St. Louis, MO), unless otherwise indicated.