Zvad fmk

Doxycycline hyclate and minocycline hydrochloride were purchased from Sigma Aldrich (Stockholm, Sweden) and COL-3 was generously provided by CollaGenex Pharmaceuticals Inc (Newtown, PA, USA) and Galderma R&D (Sophia-Antipolis, France). Stock solutions of DOXY and MINO were prepared in distilled water (dH₂O) in a concentration of 5 mg/ml and stocks of COL-3 in dimethyl sulfoxide (DMSO; Sigma Aldrich, Stockholm, Sweden) in a concentration of 50 mg/ml and stored at −20°C.
The other materials were purchased as follows: Z-VAD-FMK (Bachem AG, Bubendorf, Switzerland); etoposide (Bristol-Myers Squibb, Bromma, Sweden); RPMI 1640 medium, Dulbecco’s phosphate-buffer saline (PBS) and fetal bovine serum (FBS) (Invitrogen AB, Stockholm, Sweden); resazurin (R&D System, Stockholm, Sweden); cOmplete mini protease inhibitor cocktail (Roche AB, Stockholm, Sweden); propidium iodide (Sigma Aldrich) and annexin V (BD, Stockholm, Sweden). All other chemicals and solvents were of analytical grade and purchased from Merck, Darmstadt, Germany.

Enteroid monolayers were prepared as described previously (Thorne et al., 2018 (link)), in short, single cell suspensions were generated from organoids by mechanical disruption and a digest with TrypLE Express (Thermo Fisher Scientific Cat# 12605-010). Approximately 4000 WT and/or 1000 cancer cells were seeded per well of BME2 coated (0.8mg/ml) 96-well plate in medium supplemented with CHIR-99021 and Y-27632. After 24hrs, cells were washed once and cultured in murine small intestinal organoid medium for the remainder of the experiment. For imaging purposes cells were plated in glass-bottom 96 well SensoPlates (Greiner Bio-One Cat#655892). Small molecule inhibitors were used in the following concentrations: Z-VAD-FMK (50 μM, Bachem Cat# N-1510.0005), CHIR-99021 (3 μM, Tocris Cat#4423), Y-2763 (10 μM, Abmole Cat# M1817).

3D mixed organoid cultures were prepared as follows; suspensions of small clumps of cells were generated from organoids by mechanical disruption and divided over Eppendorf vials in a 2:1 ratio (WT:cancer). Cells were concentrated by mild centrifugation, the pellet as resuspended in a small volume of murine small intestinal organoids medium and incubated at 37C for 30 minutes. Cell aggregates were plated in BME2 and cultured in murine small intestinal organoids medium. For imaging purposes cells were plated in ibiTreat #1.5 polymer coverslip 96 Well μ-Plate (IBIDI, cat# 89626) or μ-Slide 8 Well chambered slides (IBIDI, cat#80827). Small molecule inhibitors were used in the following concentrations: Z-VAD-FMK (50 μM, Bachem Cat# N-1510.0005), CHIR-99021 (3 μM, Tocris Cat#4423), Valproic acid (1mM, Sigma Cat# PHR1061-1G), Y-2763 (10 μM, Abmole Cat# M1817), JNK-IN-8 (1 μM, Sigma Aldrich, Cat#SML1246), Doxycycline (4.25 μM, Sigma Aldrich, Cat#D9891).

Recombinant human TRAIL (375-TEC) was purchased from Bio-Techne (Wiesbaden-Nordenstadt, Germany), TNF-α from Merck (Darmstadt, Germany), zVAD.fmk from Bachem (Bubendorf, Switzerland), SC-514 from Bio-Techne (Wiesbaden-Nordenstadt, Germany), PD-0325901 from Selleckchem (Houston, Texas, USA) and Bay 11-7082 from Invivogen (San Diego, California, USA). Cell culture media and buffers were from Thermo Fisher Scientific (Darmstadt, Germany).

Cells were preincubated for 2 h with either 2 mM glutathione (Sigma-Aldrich, Munich, Germany), 50 µM zVAD-fmk (Bachem, Bubendorf, Switzerland), 5 µM Oligomycin A (BIOZOL, Eching, Germany), 2 mM ascorbic acid (Sigma-Aldrich), and 300 µM H₂O₂ (Otto Fischar GmbH, Saarbrücken, Germany). Then, cisplatin was added at 10 µM. Cells were simultaneously incubated with cisplatin and 100 µM Genipin (Sigma-Aldrich).

To estimate the effect of DBeQ on this functional response, we used a bioassay previously optimized by our group [55 (link), 56 (link)]. As controls, we performed also assays using the agonistic anti-Fas mAb CH11 (Upstate, Lake Placid, USA) at 50 ng/ml or recombinant TRAIL produced in our laboratory (e.g., see ref. 31) at 1 μg/ml, in the presence or absence of 3 μM DBeQ and/or the general caspase inhibitor Z-VAD-fmk (Bachem, Bubendorf, Switzerland) at 30 μM. For supernatant testing, Jurkat cells or T cell blasts were preincubated or not during 16h with 3 μM DBeQ, and, after washing out the inhibitor, cells were stimulated with 10 ng/ml of phorbol myristate-acetate (PMA) plus 600 nM ionomycin during 2h. Cell supernatants of stimulated cells were recovered by centrifugation, and tested overnight against non-activated Jurkat cells, as indicated in [56 (link)]. Cell death was estimated in these experiments by nuclear staining of dead cells with 7-amino-actinomycin D (7-AAD) and flow cytometry. In our previous studies we have shown that cytotoxicity on Jurkat cells of these supernatants is mainly due to FasL and Apo2L/TRAIL secretion associated with exosomes [8 (link), 56 (link), 57 (link)], being thus a functional test of exosome secretion.