Fetal calf serum (fcs)

Bone marrow was aspirated bilaterally from both the anterior and posterior iliac crests (10 mL/site) from breast cancer patients or healthy volunteers. The following procedures were accomplished under sterile conditions. The bone marrow aspirates were washed in Hanks’ Balanced Salt solution (HBSS) (Biochrom AG, Berlin, Germany), diluted in Dulbecco’s phosphate-buffered saline (DPBS) (Life Technologies), and separated by density centrifugation using Ficoll Paque Plus (GE Healthcare, Munich, Germany). Mononuclear cells were collected from the interphase layer and washed twice in phosphate buffered saline (PBS) with 10% fetal calf serum (Biological Industries, Kibbutz Beit Haemek, Israel). The cytospins were prepared by centrifuging the bone marrow mononuclear cells onto glass slides (Superfrost plus, Glaswarenfabrik Karl Hecht KG, Sondheim, Germany; 7 × 10⁵ mononuclear cells per slide). The slides were air-dried overnight and stored at −80 °C.
For the experiments using peripheral blood from healthy individuals, the PBMC were separated by Ficoll density centrifugation. The obtained PBMC were collected from the interphase layer, washed with PBS, and either applied for the spiking experiments or analyzed by Western blot analysis. For the spiking experiments, the cell lines were spiked into the blood or bone marrow from healthy individuals and processed as described.

Rat pancreatoma AR42J cells (American Type Culture Collection, MD, USA) were maintained as a sub-confluent monolayer culture in Dulbecco’s Eagle’s medium (Rhenium, Jerusalem, Israel) containing 10% (v/v) fetal calf serum (Biological Industries, Beit Haemek, Israel) and 1% (v/v) penicillin–streptomycin. Cells were grown in 5% CO₂ at 37°C. Dexamethasone (100 nM; Sigma-Aldrich, Rehovot, Israel) was added to the cells for 48 h before the experiments to induce cell differentiation. Tunicamycin (TM, 5 µg/ml) was used as appositive ER stress inducer (Sigma-Aldrich, Rehovot, Israel). CER (10 nmol/l) (Sigma-Aldrich, Rehovot, Israel) was added as stress inducer.

Dulbecco’s modified Eagle’s medium (DMEM) supplemented with L-glutamine was purchased from Gibco (Grand Island, NY). Fetal calf serum, penicillin-streptomycin, sodium pyruvate and non-essential amino acids were purchased from Biological Industries (Beit Haemek, Israel) Anti-actin monoclonal antibodies were from Millipore (Billerica, MA). Goat polyclonal anti-HK-I (sc-6517) and anti-HK-II (sc-6521) antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-BAX (PC66) and mouse monoclonal anti-Bcl-2 (OP60) antibodies were obtained from Calbiochem (Billerica, MA). Rabbit polyclonal anti-AIF (AF1457) antibodies were purchased from R&D Systems (Minneapolis, MN). Rabbit polyclonal anti-MAVS (ab-56230), rabbit polyclonal anti-VDAC1 (ab15895), rabbit polyclonal anti-SMAC/Diablo (ab-8115), anti-PPWD1 (ab-126710), anti-SLC25A1 (ab-99168), anti-DHRS4 (ab-68095), anti-UBE3A (ab-126765) and Cy2-conjugated anti-rabbit antibodies were obtained from Abcam (Cambridge, UK). Monoclonal anti-CD19 and CD5 antibodies were obtained from BD Bioscience (San Jose, CA). Horseradish peroxidase (HRP)-conjugated anti-mouse, anti-rabbit and anti-goat antibodies were from KPL (Gaithersburg, MD).

Whole blood was lysed with RBC lysing solution (BioLegend). Cells were washed and the cell count was performed by MACSQuant cytometer. Cells were resuspended in RPMI 1640 with 10% fetal calf serum (Biological Industries, Kibbutz Beit Haemek, Israel), 2 mM glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin (Biowhittaker, Verviers, Belgium) at 2 × 10⁶ cells/ml. In 24 plate-wells, 500 μl of RPMI 1640 with 10% fetal calf serum or 0.22 μm filtered medium RPMI supplemented with 10% of pleural fluid from LAC was added to the wells. 3 × 10⁵ cells were added in 3 μm pore-size filters (Millipore Corporation, Billerica, MA) and incubated for 4 h at 37ΊC. After culture, healthy donor cells that migrated towards the medium or pleural fluids were collected from the wells, washed with PBS and stained with anti-CD3-PECy5, CD4-PECy7 (BioLegend) and CD20-FITC (Immunotools) monoclonal antibodies for 20 min. Cells were washed and resuspended in 300 μl of PBS to be acquired by flow cytometry. To analyze the migration fold of cells towards the pleural fluids, we calculated the relative cell migration that is the ratio between the absolute numbers of CD4+ T, CD8+ T cells and CD20+ B cells that migrate to the medium supplemented with pleural fluid and the absolute cell numbers of each respective subset that migrate to the medium.

Phosphate buffered saline (PBS), Dulbecco's Modified Eagle's Medium (DMEM), fetal calf serum (FCS), L-glutamine, antibiotics combination of streptomycin and penicillin, and Trypsin-EDTA were all purchased from Biological Industries (Beit Haemek, Israel). BSA and Tween-20 were purchased from ICN Biomedicals, Inc. (Aurora, OH, USA).

U2OS cells were grown at 37 °C and 5% CO₂ in DMEM supplemented with 10% Fetal calf serum (FCS, Biological industries, Beit Haemk, Israel), 4.5 g/L glucose, 2 mM L-Glutamine, 20 mM HEPES-KOH pH 7.4, 100 U/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml amphotericin B. Starvation conditions: Cells were rinsed twice with PBS and incubated in starvation medium (Glucose free DMEM supplemented with 20 mM HEPES-KOH pH 7.4, 5 mg/ml BSA, 100 U/ml penicillin, 100 µg/ml streptomycin and 0.25 µg/ml amphotericin B) for two hours. Antioxidants were prepared according to the manufacturer’s directions and diluted in media prior to being added to the cells.