Apo transferrin

Apo-transferrin, biotin, Bradford solution, cortisol, dexamethasone, dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Dulbecco’s modified Eagle’s medium/nutrient F-12 Ham, fetal bovine serum, 4% formalin solution, insulin, 3-isobutyl-1-methylxantine, isopropanol, Oil red O solution (ORO), pantothenic acid, penicillin/streptomycin 10 000 IU/10 mg/ml, protease and phosphatase inhibitor cocktail, RIPA lysis buffer, rosiglitazone, triiodothyronine and 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TSPA-d4; purity 99%) were supplied from Merck KGaA (Darmstadt, Germany). Pure AST (purity ≥ 95%) and QUE (purity ≥ 95%) were purchased from PhytoLab GmbH and Co. KG (Vestenbergsgreuth, Germany). Deuterated methanol (CD₃OD; 99.8%) and water (D₂O; 99.9%) were purchased by Deutero GmbH (Kasbellaun, Germany). All chemicals and reagents for electrophoresis, immunoblotting and real-time quantitative reverse transcription-polymerase chain reaction (RT-qPCR) were purchased from Bio-Rad Laboratories Inc. (Hercules, CA, United States).

At first, apo transferrin (10 mg, Merck, Rahway, NJ, United States) was dissolved in 2 mL of DPBS (pH = 6.8). Fluorescein-6 maleimide (Lumiprobe, Russia) in the amount of 2 µL (20 mg/mL in DMSO) was added and mixed overnight at constant stirring at ambient temperature. After that, the resulting solution was purified five times using centrifugal concentrator 30 kDa cut off filters (Millipore Billerica, United States) and adjusted to 1 mL in PBS (pH = 7.4). Then, 10 μL of 6-Maleimidohexanoic acid N-hydroxysuccinimide ester at concentration of 0.5 mg/mL in water:DMSO 1:1 was mixed with Tf-FAM followed by 1 h incubation at ambient temperature. The solution was also purified five times by 30 kDa cut off filters and resolved in 1 mL PBS (pH = 6.8). At last, the isolated Mx encapsulins were also diluted in PBS at pH = 6.8 and mixed 1:1: by weight in 1 mL of the reaction mixture overnight. The produced conjugates were washed seven times using 100 kDa cut off filters and PBS (pH = 7.4). The 500 ng/μL Mx-Tf-FAM stock solution was kept at 4°C.

The Institutional Review Board of Yeungnam University Medical Center (IRB No: 2019-04-047 at 21 December 2019) approved HPMC collection using human specimens. Briefly, gastrectomy was considered for all biopsy-confirmed patients with stomach cancer and was performed using partial or total gastrectomy with total or partial omentectomy. The remnant omentum is typically discarded at our center after surgery. Therefore, prior to surgery, patient informed consent was obtained for use of mesothelial cells from the discarded omentum. HPMCs were isolated as previously described [12 (link),13 (link),14 (link)]. In vitro experiments were performed on cells after one to two passages. HPMCs were cultured in M199 medium supplemented with L-glutamine (20 μM, Gibco, Waltham, MA, USA), penicillin-streptomycin (150 units or 150 μg/mL, Gibco), hydrocortisone (0.4 μg/mL, Sigma-Aldrich, St. Louis, MO, USA), apotransferrin (5 μg/mL, Sigma), insulin (5 μg/mL. Sigma) and 10% fetal bovine serum (FBS) (Gibco). Cell cultures were maintained in 100 mm dishes (Nunc, Rochester, NY, USA) in a 5% CO₂ humidified atmosphere incubator (311 Forma Direct Heat CO₂ incubator, Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C.

EMEM growth medium, fetal bovine
serum, penicillin–streptomycin,
and supplements (glutamine and pyruvate) were purchased from Biological
Industries (Beit-Haémek, Israel). Apo-transferrin and PBS pH
7.2 were from Sigma Aldrich. The materials used for synthesis and
work-up procedures were purchased from Sigma Aldrich, Merck, Fluka,
and Frutarom and used upon arrival unless otherwise stated. Deuterated
solvents (Sigma Aldrich isotopes products) with a 99.5% minimum deuteration
were used upon arrival. Silica gel for column chromatography (Silica
Gel 60, 63–200 μm mesh) was obtained from E. Merck Ltd.
Pyrrole was run through a short basic alumina column, and aldehydes
were purified by vacuum distillation before use. HPLC analysis was
performed on a combination of a JASCO organizer, a diode array detector
MD-4010, an autosampler AS-4050, and an RHPLC pump PU-4180. The silica
gel (230–400 mesh) used for column chromatography was obtained
from E. Merck Ltd. A purity of >95% for 3-Ga was determined
with HPLC and UV detection at their Soret band region; a summary of
HPLC results and the HPLC conditions is shown in the Supporting Information.

The U5 fetal human NSC line (Bressan et al, 2017) and adult 0131‐mesenchymal and 0827‐proneural human GSC lines (Son et al, 2009) were grown in NeuroCult NS‐A basal medium (StemCell Technologies) supplemented with B27 (Thermo Fisher), N2 (2× stock in Advanced DMEM/F‐12 (Fisher) with 25 μg/ml insulin (Sigma), 100 μg/ml apo‐Transferrin (Sigma), 6 ng/ml progesterone (Sigma), 16 μg/ml putrescine (Sigma), 30 nM sodium selenite (Sigma), and 50 μg/ml bovine serum albumin (Sigma), and EGF and FGF‐2 (20 ng/ml each) (Peprotech) on laminin (Sigma or Trevigen)‐coated polystyrene plates and passaged according to previously published protocols (Pollard et al, 2009). Cells were detached from their plates using Accutase (Thermo Fisher). 293T (ATCC) cells were grown in 10% FBS/DMEM (Invitrogen).

BCC shave biopsies were obtained from the dermatology department of the university hospital Zürich (USZ). Only material from consenting patients was used for this purpose. After collection, one shave biopsy was cut into 2 pieces, 1 piece was put directly in cell culture insert (Millipore, Cat. No. Z352985), and the other part slow-frozen. The insert was placed in a 6-well plate and the shave biopsy on the insert was cultivated for 5 days with 1.5 ml of co-culture medium only at the bottom. The medium was changed every second day. After 5 days, IHC was performed to measure proliferation (Ki67) and the presence of BCC cells (BerEP4). The co-culture medium is composed as follows: 3 parts DMEM (Gibco, Cat. No. 41966052), 1 part Ham’s F-12 Nutrient Mix (Gibco, Cat. No. 11765054), 10% Fetal Bovine serum heat inactivated (Gibco, Cat. No.1050), 0.1 mg/ml of Normocin^TM (Invivogen, Cat. No. ant-nr-1), 21.8 μg/mL of Adenin (Sigma-Aldrich, Cat. No. A2786), 5.45 μg/mL of Apotransferrin (Sigma-Aldrich, Cat. No. T1147), 2.18 nM of Triiodothyronine (Sigma-Aldrich, Cat. No. T6397), 0.44 μg/mL of Hydrocortisone (Sigma-Aldrich, Cat. No. H0888), 0.11 nM of Cholera toxin (Sigma-Aldrich, Cat. No. C8052), 5.50 μg/mL of Insulin (Sigma-Aldrich, Cat. No. I6634) and 0.01 μg/mL of Epidermal growth factor (Sigma-Aldrich, Cat. No. E4127)