Brefeldin a

Brefeldin A, SCH772984, 4μ8C, KIRA6, and mizoribine were purchased by Selleckchem. BAY11–7082, GSK2606414, STF-083010 were purchased by Caymanchem). N-Acetyl-L-cysteine and L-Histidine were supplied by Sigma–Aldrich, thapsigargin by Enzo Life Sciences, BAPTA-AM by ThermoFisher Scientific. Bortezomib was obtained directly from the Pharmacy Department of the Leuven University Hospital, Leuven Belgium.

Verification of BiP cleavage in the intoxicated HeLa cells was performed with 1 × 10⁶ cells in 12.5 mL, seeded in 12-well plates distributed to 8 × 10⁴ cell per well. After overnight growth, the cells were washed one time with PBS and incubated with serum-free MEM or 10 µmol/L Brefeldin A (Selleckchem, Munich, Germany) in serum-free MEM for 30 min. Subsequently, the toxin was added, and the cells were further incubated for 4 h. In total, 10 µg/mL toxin was added to the cells containing the A- and B-subunit in a 1:5 molar ratio. After another washing step with PBS, cells were detached by adding 2.5× Laemmli sample buffer containing DTT and mechanically removed from the surface. Cells were transferred in reaction tubes to be incubated at 95 °C for 10 min. Then, 40 µL of each sample was applied to a 12.5% SDS PAGE. Following this, the proteins were blotted onto a nitrocellulose membrane, and BiP was detected using anti-GRP78/BiP rat (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA) and anti-rat-HRP (1:2500, Cell Signaling Technology, Danvers, MA, USA). The detection was performed by enhanced chemiluminescence (ECL, Millipore, Burlington, MA, USA). For loading control, Hsp90 was detected after stripping of the blot using anti-Hsp90-mouse (1:1000, Santa Cruz, Dallas, TX, USA) and mBP (1:2500, Santa Cruz, Dallas, TX, USA).

U87, RADH85 and RADH87 (WT, DN, EV, IRE1WT or IRE1Q780*) and MDA-MB-231 WT or CD95 KO1 and 2 (initially named CD95 KO 5 and KO 9 respectively) were generated in our laboratory as described previously (Avril et al, 2012 (link); Guégan et al, 2021 (link); Lhomond et al, 2018 (link); Nguyên et al, 2004 (link); Pluquet et al, 2013 (link)). All cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% decomplemented FBS and 2 mM L-glutamine at 37 °C in a 5% CO₂ incubator. Modified RADH85 and 87 were cultured with 0.8 μg/mL or 1 μg/mL puromycin, respectively. All cells were regularly tested for mycoplasma absence. CD95L was produced and quantified in-house as described previously (Risso et al, 2023 (link)). Thapsigargin (SML1845), Actinomycin D (A9415) and Tunicamycin (T7765) were from Sigma-Aldrich. Brefeldin A (S7046), MKC-8866 (S8875), Birinapant (S7015), MG-132 (S2619) were from Selleckchem, Z4 was from Enamine (Z940452448).

In HVHF group, patients were connected to the blood filtration machine within 6 hours after admission, and venous blood samples (5 mL) were collected at 0 hours (before HVHF), 6, 12, and 24 hours. Samples (5 mL) from non-HVHF patients were collected at equivalent time points during routine treatment within 6 hours of admission. During the same period, isovolumetric blood samples were drawn from healthy volunteers in the physical examination center of our hospital. All samples were refrigerated at 4°C after EDTA anticoagulation. Serum and peripheral blood mononuclear cells were separated by density gradient centrifugation at 2000 rpm for 20 minutes. The former was stored at −80°C for subsequent cytokine detection and the latter was adjusted to 1 × 10^6–8/mL in RPMI 1640 medium supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM/L glutamine, and 10% fetal calf serum (Gibco BRL). The cell suspension was then seeded into 24-well cell culture plates. Cells were treated with phorbol myristate acetate (Abcam; 50 ng/mL), ionomycin (Abcam; 2 g/mL), and brefeldin A (Selleck Chemicals, Houston, TX; 3 g/mL), and incubated in the dark at 37°C under 5% CO₂ atmosphere for 5 hours.