Mercaptoethanol

We utilized human melanoma cell lines NW-Mel-38 and SK-Mel-37 as cell lines that express the MAGEA4 target antigen. For our experiments, we used the HCT-116 cell line as a negative control, which does not express the MAGE-A4 target antigen. The tumor cell lines were cultivated in culture flasks with a planting concentration of 50–75 thousand cells/mL until they reached the log-phase of growth. RPMI-1640 medium supplemented with 10% FCS (Hyclone, Logan, UT, USA), 2 mM L-glutamine (Biolot, Russia), 5 × 10⁻⁵ mM mercaptoethanol (Sigma, Cibolo, TX, USA), 25 mM HEPES (Sigma, USA), 80 µg/mL gentamicin (Krka, Novo Mesto, Slovenia), and 100 µg/mL ampicillin (Sintez, Kurgan, Russia) were used for cell culture. After detachment from the plastic surface using trypsin-versen solution (Biolot, St. Petersburg, Russia), the cells were seeded into the wells (4–5 × 10⁴ cells/well) of a 96-well flat-bottomed culture plate (TPP, Switzerland) for 16–17 h, ensuring proper adhesion of the tumor cells to the plastic. The cell lines NW-Mel-38 and SK-Mel-37 were obtained from the collection of cell cultures at the Graduate School of Medicine, Mie University, under the supervision of Professor H. Shiku.

The samples were immersed in 350 µl lysis buffer (RLT solution; Qiagen, Germany) plus 3.5 µl mercaptoethanol (Sigma), and then pulled ten times through a 22-gauge needle. Total RNA was obtained from individual MV samples or RT and BT with RNeasy® Plus Micro kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The concentration and quantity were determined using the Agilent 6000 Pico kit (RMVs and BMVs), or Agilent Nano kit (brain and retinal tissues) on an Agilent 2100 bioanalyzer (Agilent Technologies, Waldbronn, Germany). Only samples with an integrity number (RIN) > 8.0 were used for microarray processing.

For structured illumination microscopy (SIM) image acquisition, labeled cells on glass coverslips were mounted with Fluoroshield™, containing DAPI (Thermo Fisher). Images were recorded with the 40 × alpha 1.46 Plan apochromat objective with oil immersion (Zeiss, Oberkochen, Germany). Z-stacks were recorded in SIM mode with a 16 bit depth at 5 angles, with averaging 4; 51 µm grid was applied for 633 laser line. The acquired SIM dataset were reconstructed by the ZEN software (Zeiss).
To perform PALM imaging, labeled cells were kept in an imaging solution containing 10% Glucose, 10 mM sodium chloride, 50 mM Tris–HCl, catalase, Pyranose oxidase, 100 mM Cysteamine, 2 mM Cyclooctatetraene and 100 mM Mercaptoethanol (all Sigma Aldrich). The detailed process of image acquisition was reported previously [21 (link)]. A total of 5000–10,000 frames were acquired using a Zeiss ELYRA LSM 780 imaging system (Zeiss) equipped with a 1.57 N.A. 100 × oil objective. Subsequently, reconstruction of raw images and post-processing was performed with Image J software and the Thunderstorm plugin [22 (link)].

Proteins of whole brain lysates were analyzed by immunoblotting against β1i/LMP2 gp, β5i/LMP7 rb (both custom-generated), β2i/MECL-1 [K65 rb; (27 (link))] and β-Actin (#A1978, Sigma-Aldrich).
Tibias and femurs of 10-14 weeks-old WT and TKO mice were aseptically removed, and bone marrow cells were flushed out with sterile PBS and centrifuged at 150 ×g for 10 min. Cells were resuspended in RPMI medium containing 10% FCS (Capricorn), recombinant murine granulocyte-macrophage colony-stimulating factor (2 ng/ml; Cell Signaling Technology) and 50 μM mercaptoethanol (Sigma-Aldrich) and cultivated for at least 10 days at 37 °C and 5% CO₂. Twenty-four hours prior to experiments, cells were harvested by scraping and seeded into 6-well plates. For investigation of signaling events cells were treated for the depicted time points with 30 µg/ml Toxoplasma lysate Antigen (TLA) and harvested using Trizol reagent (Invitrogen). Proteins were quantified via Bradford assay and subsequently analyzed by immunoblotting against pStat3 (Tyr705) (D3A7; XP^® Rabbit mAb #9145 CST), Stat3, pMEK (Ser217/221) (41G9; Rabbit mAb #9154 CST), pErk (Thr202/Tyr204) (20G11; Rabbit mAb #4376 CST), Erk and GAPDH (all Cell Signaling Technology) antibodies.

M199 medium, gelatin, penicillin, streptomycin, heparin, NaOH, sodium dodecylsulfate (SDS), Tris, phosphate-buffered saline (PBS), ethylendiaminetetraacetic acid (EDTA), chloroform, glycerol, trichloroacetic acid, trypsin, hydroxyurea, glucose, bromophenol blue, lipofectamine, CO-releasing molecule-2 (CORM2), NADPH, glucose-6-phosphate, glucose-6-phosphate dehydrogenase, Triton X-100, and mercaptoethanol were from Sigma-Aldrich (St. Louis, MO, USA). Bilirubin and tin protoporphyrin were from Frontier Scientific (Logan, UT, USA). The antibody against HO-1 was from Assay Designs (Ann Arbor, MI, USA) and the antibody against β-actin was from Santa Cruz (Santa Cruz, CA, USA). Tumor necrosis factor-α (TNFα) was from R&D Systems (Minneapolis, MN, USA). Canagliflozin was purchased from Selleck Chemicals (Houston, TX, USA). [³H]Thymidine was from Perkin Elmer (Boston, MA, USA).