Mem amino acid solution

YCCEL1 cells were washed with PBS and then starved by incubation in amino acid-free EMEM with 10% dialyzed FBS (Gibco) at 37°C for 50 minutes. Amino acid-free EMEM was prepared based on formulation of ATCC 30–2003. Amino acid stimulation was then performed by adding 100X MEM amino acid solution (1:100, ThermoFisher) at 37°C for 10min.

Microvessels were fabricated in collagen and hylauronan (HA) composite hydrogels polymerized inside polydimethylsiloxane (PDMS)-based microfluidic devices fabricated using soft lithography^{43 (link)}. p20–p23 human cerebral microvascular endothelial cells (hCMEC/D3) and p7–p15 GFP-labeled human brain vascular pericytes (hBVP) (Neuromics) were seeded in the hydrogels at densities of 2 M/mL and 0.4 M/mL, respectively. These ratios were obtained from a previous study that used co-cultures of human blood outgrowth endothelial cells and human pericytes to form microvasculature networks^{13 (link)}. hCMEC/D3 were cultured in Endothelial Cell Basal Medium (PromoCell) supplemented with 5 µg/mL ascorbic acid (Sigma), 1 ng/mL hBFGF (Sigma), 1/100 chemically defined lipid concentrate (Thermo Fisher), 5% fetal bovine serum (VWR Life Science), 10 mM HEPES (Quality Biological), 1.4 µM hydrocortisone (Sigma), and 1% penicillin-streptomycin (Corning). hBVP were cultured in DMEM (Corning) supplemented with 10% FBS, 1% penicillin-steptomycin, and 1X MEM Amino Acid Solution (Thermo Fisher). Final hydrogel formulation concentrations consisted of 3 mg/mL HA (Sigma), 5 mg/mL collagen type I (MP Biomedical), and 0.85–1 mg/mL Matrigel (Corning). These reagents were combined with 0.1 M sodium hydroxide (NaOH) and 10x phosphate buffer solution (PBS) to facilitate polymerization and maintain physiological pH.

HepG2 cells were purchased from the American Type Culture Collection (ATCC) and cultured in 10% serum-containing DMEM as previously described [66 (link)]. Mouse embryonic fibroblasts (MEFs) eIF2α S/S and MEFs eIF2α A/A were prepared and cultured as described previously [67 (link)]. MEFs (eIF2α S/S and eIF2α A/A) were cultured in 10% serum-containing DMEM medium containing MEM amino acid solution (50×, Gibco) and MEM non-essential amino acid solution (100×, Gibco).

A. phagocytophilum isolate NY-18 [17 (link)] (kindly provided by Prof. José de la Fuente, University of Castilla-La Mancha, Ciudad Real, Spain) was propagated in HL-60 promyelocytic cell line (ATCC, CCL-240) in RPMI 1640 culture medium (GIBCO) containing 2% MEM amino acid solution (GIBCO) and 10% heat-inactivated (56 °C, 30 min) fetal calf serum (FCS, Lonza) at 37 °C with 5% CO₂ [29 (link)]. The number of infected cells was monitored under an Olympus BX53 microscope. The cell smear was air-dried and stained with RAL DIFF-QUIK (Siemens Healthineers, Erlangen, Germany) according to the manufacturer’s instructions. Cultures were injected into mice when ~ 50% of cells were infected.

The strains used in the paper were derivatives of Escherichia coli (E. coli) K‐12 which were bought from the E. coli Genetic Stock Center^[30 (link)
^] (Yale University, New Haven). The observed target gene was cspC, a member of cold shock proteins, with the function of up‐regulating the level of RNA polymerase. The CAT gene was located downstream of each YFP fusion behind its own constitutive promoter, the whole strain could be expressed as cspC‐YFP(::cat). The bacteria cells were first cultured in LB from a single colony picked on an agar plate for 6–7 h, then they were transferred into M9 minimal medium supplemented with glucose (0.4%) and amino acids (2%, 50 × MEM amino acid solution, Gibco) shaking at 250 rpm under 37 °C and cultured for overnight. Small gel pads with ≈2% agarose (Sigma) were prepared through heating and placed between the microslide and coverslip of the FSC2 to immobilize the cells from moving. Then 0.4 µL of the bacteria solution was injected into the pads. Fresh M9 minimal medium with different Cm concentrations was supplemented to the culture area in real‐time with a peristaltic pump. Cm stock solution (20 mg mL⁻¹, Aladdin) dissolved in 70% isopropanol solution was prepared before dilution into 0.2–1.0 mm in M9 medium.

Hepatoma HepG2 cells were maintained in DMEM (GE Healthcare, Buckinghamshire, UK) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 5% CO₂. Unless indicated otherwise, cystine-free medium was prepared by supplementing cystine-, methionine- and glutamine-free DMEM (Gibco, CA, USA) with 100 μM methionine, 4 mM glutamine, 1 mM sodium pyruvate, 0.2% bovine serum albumin (BSA), 100 U/ml penicillin and 100 μg/ml streptomycin. The control medium contained 100 μM cystine. For cell viability assays, medium was supplemented with 10% dialyzed FBS. For GSH assay, cells were treated in Earle’s balanced salt solution (EBSS), supplemented with 25 mM glucose, 4 mM glutamine, 1 X MEM vitamin solution (Life technologies, CA, USA), 0.22% wt/vol NaHCO₃, 1 mM sodium pyruvate, 0.2% BSA, 25 mM HEPES, 100 U/ml penicillin and 100 μg/ml streptomycin in the presence or absence of 1 X MEM amino acid solution (Gibco, CA, USA) with or without 100 μM cystine. For the measurement of ROS and lipid peroxidation, cells were treated in phenol red-free DMEM with 10% dialyzed FBS, 100 U/ml penicillin and 100 μg/ml streptomycin with or without cystine.