L serine

Human glioblastoma cell line A172 and SF767 were respectively cultured in DMEM medium (Gibco^®, Invitrogen) supplemented with 10% fetal bovine serum without or with 1X MEM Non-Essential Amino-Acids containing Glycine 75 mg/L, L-Alanine 89 mg/L, L-Asparagine 132 mg/L, L-Aspartic acid 133 mg/L, L-Glutamic acid A 47 mg/L, L-Proline 115 mg/L, L-Serine 105 mg/L (Gibco^®, Invitrogen). A172/SF767 GBM cells were transfected using Interferin (Polyplus Transfection) according to the manufacturer's recommendations. Briefly, cells were plated in six-wells tissue culture plates at a density of 1.10⁵ and transfected 24 hours later with 10 nM of siRNA control (non-targeting 2, Dharmacon), 10 nM of siEFBN3 and 2.5 nM of siEPHA4. 24 hours later, cells were transfected a second time with 10nM of siRNA control (non-targeting 2, Dharmacon) or targeting EFBN3.

Immortalized gingiva keratinocyte (KC-TERT, OKG4/bmi1/TERT, Rheinwald laboratory, Boston, USA) (passage 10 to 20) and fibroblast (Fib-TERT, T0026, ABM, Richmond, BC, Canada) (passage 10 to 20) cell lines were used for constructing RHG. The RHG model, consisting of a fibroblast-populated collagen hydrogel (collagen I derived from rat tail) overlaid with keratinocytes (0.5 x 10⁶ cells) was cultured exactly as previously described (Kosten et al., 2015 (link)). In brief, RHG were cultured in six-well transwell inserts (pore size 0.4μm, 24mm, Corning, USA), first submerged in culture medium for 3 days and then lifted to the air-liquid interface for an additional 10 days to induce epithelial differentiation and stratification in culture medium containing DMEM/Ham’s F12 (3/1) (Gibco, Grand Island, USA) supplemented with 1% Fetal Clone III (HyClone, GE Healthcare, Chicago, USA), 1% PS (Gibco, Grand Island, USA), 0.1µM insulin, 1µM hydrocortisone, 1µM isoproterenol, 10µM L-carnitine, 10mM L-serine, 0.4mM ascorbic acid, and 2ng/mL epidermal growth factor. All agents were purchased at Sigma-Aldrich (St. Louis, USA) when not specified. One day prior to bacteria exposure, PS and hydrocortisone were omitted in RHG culture medium for some experimental conditions as indicated below.

Penicillin–streptomycin 100,000 UI/mL, poly(ethylenimine) (PEI) 1300~50% in H₂O, sodium citrate, cytochrome c from equine heart, transferrin, fibrinogen, human serum albumin, bovine serum albumin, staurosporine from streptomyces, and 3-(4,5-dimethulthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich, St. Louis, MO, USA. Slide-A-Lyzer^® Dialysis Cassette–2000 MWCO 3.0–12 mL Capacity and DRAQ5™ were purchased from Thermo Scientific, Rockford, IL, USA. l-Valine, l-Serine, l-Methionine, l-Leucine, l-Aspartic Acid, l-Tryptophan, l-Asparagine monohydrate, l-Phenylalanine were purchased from ThermoFischer (Kandel, Germany). Fetal bovine serum and phosphate saline buffer were purchased from Gibco, Grand Island, NY, USA. Etoposide was purchased from Fresenius Kabi, Bad Homburg, Germany. CytoPainter Mitochondrial Staining Kit Red Fluorescence was purchased from Abcam, Cambridge, UK. Phalloidin-FITC Acti-stainTM 488 Fluorescent Phalloidin was purchased from Cytoskeleton Inc., Denver, CO, USA. Prolong^® Gold Antifade Reagent Molecular Probes was purchased from Cell Signaling Technology, Danvers, CO, USA. Double distilled water (18.2 MΩ) was used as a solvent.

Sodium alginate from Millipore Sigma (Cat# A1112) (LV), BASF (Hydagen 558P) (HV1), TIC (Algin 400) (HV2), and DuPont Danisco (GRINDSTED Alginate FD 155) (HV3) were used in this study (Table 1). Calcium hydrogen phosphate, succinic acid, sodium citrate, glacial acetic acid, sodium carbonate, β-D-glucose, sodium bicarbonate, L-serine, sodium hydroxide, ammonium hydroxide, hydrochloric acid, calcium carbonate, and methanol were purchased from Thermo Fisher. Schiff’s fuchsin sulfite reagent, sodium metabisulfate, periodic acid, anthrone, D-(+)-galacturonic acid, concentrated sulfuric acid, disodium 2,2-bicinchoninate (BCA), copper (II) sulfate, chloroform, 1-phenyl-3-methyl-5-pyrazolone (PMP), and D-(+)-mannuronic acid were purchased from Millipore Sigma. The polymannuronic acid (YP31737) and polyguluronic acid (YP03135) was obtained from Carbosynth Ltd. (Berkshire UK) at a minimum of 85% purity and 10% water content with an average molecular weight of 6–8 kDa.

l‐Arginine (C₆H₁₄N₄O, ≥ 98%), l‐glutamic acid (C₅H₉NO₄, ≥ 99%), l‐proline, (C₅H₉NO₂, 99%), l‐asparagine (C₄H₈N₂O₃, 99%), l‐glutamine (C₅H₁₀N₂O₂, ≥ 98%), l‐aspartic acid (C₄H₇NO₄, ≥98%), and l‐threonine (C₄H₉NO₃, ≥ 98%) were bought from Sigma–Aldrich; l‐histidine (C₆H₉N₃O₂, 99%), and l‐serine (C₃H₇NO₃, 99%) were purchased from Alfa Aesar. l‐Alanine (C₃H₇NO₂, 99%), l‐cysteine (C₃H₇NO₂S, 99%), and glycine (C₂H₅NO₂, 99%) were purchased from Acros Organics. d‐aspartic acid (99%) was purchased from Sigma–Aldrich. Silk fibroin was purchased from ChemSrc, collagen (calf skin) was purchased from Merck, and gamma globulin (bovine blood) was purchased from Sigma–Aldrich. Deionized (DI) water from a Milli‐Q ultrapure system and acetone from Echo Chemical Co. were used throughout the experiments. By using high‐purity deionized water (18.2 MΩ cm⁻¹), all aqueous solutions were prepared. Without any further modification, all the chemicals were used as received. The single‐side polished highly doped p‐type (1 0 0) Silicon wafer was purchased from Twice Jin Limited, Taiwan. The thickness of the Si wafer is 675 ± 25 µm and the resistivity is 1–10 ohm‐cm.