Z vad fmk fmk001

2-Deoxy-glucose, mannose, D-(+)-Glucose, rapamycin, and fenofibrate (were purchased from Sigma-Aldrich (St. Louis, MO). General Caspase Inhibitor Z-VAD-FMK (FMK001) was from R&D Systems (MN). The following rabbit primary antibodies used were from Cell Signaling Technology (Danvers, MA): LC3B (2775 which preferentially detects LC3B-II), Grp78 (3177), CHOP (2895), pAMPKa (Thr172, 2535), p-p70S6K (Thr389, 9234), p-eIF2a (Ser51, 3597), p-4EBP1 (Thr37/46, 9459), Cleaved Caspase-3 (9664) and Mcl-1 (4572). Mouse anti-b-actin (A5441) primary antibody was from Sigma-Aldrich. Mouse anti-Noxa was from Calbiochem (OP180). Horseradish peroxidase-conjugated anti-rabbit (W4011) and anti-mouse (W4021) secondary antibody were purchased from Promega (Madison, WI).

The pancreatic cancer cells used in this study (8988T and 8902) were acquired from the American Type Culture Collection (ATCC, Manassas, VA). These cells were incubated at 37 °C in humidified air with 5% CO₂ and grown in DMEM, and RPMI 1640 medium (Costar, Corning, NY, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 ug/mL streptomycin (Hyclone, Pittsburgh, PA, USA). All cells were routinely tested for mycoplasma contamination. Vitamin C was acquired from Sigma-Aldrich (St. Louis, MO, USA). CHIR99021 was obtained from MedChemExpress, and zVad-fmk (FMK001) was purchased from R&D systems (R&D systems, Minneapolis, MN, USA).

Galeterone, DHT, VNPT-178, VNLG-74A, and abiraterone were synthesized in-house as previously described [7 (link), 35 (link)]. Enzalutamide (a.k.a. MDV3100, #SRP016825m) was purchased from Sequoia Research Products (Pangbourne, UK). Methyltrienolone (a.k.a. R1881, #965M935) was purchased from OChem Incorporation (Des Plaines, IL). Z-VAD-FMK (FMK001) was purchased from R&D Systems (Minneapolis, MN). Etoposide (E1383) was purchased from Sigma (St. Louis, MO). Calpeptin (SC-202516) was purchased from Santa Cruz Biotechnology (Dallas, TX). All compounds were solubilized in 95% ethanol (Pharmco-AAPER; Brookfield, CT) or ultrapure DMSO (AmericanBio; Natick, MA).

MDA-MB-231 cells were cultured to a confluency of ca. 80% on either 6-well or 12-well plates, depending on the experiment. The cells were incubated in a humidified incubator at 5% CO₂ and 37 °C for 24–48 h. Subsequently, the media was changed to FBS supplemented DMEM without penicillin–streptomycin, and different concentrations of Magnetospirillum magneticum AMB-1 were added into the wells. Unless specified otherwise, the plates were always placed in a sealable box which was subsequently flushed with nitrogen for 15 min to produce hypoxic conditions. The plates were incubated using this setup at hypoxic conditions at 37 °C for 24–48 h. Using phenol red as pH indicator, the color of the media was monitored throughout the experiment, and a pH in the order of ~ 7.5 was ensured. To benchmark the iron-chelating potential of bacteria against known agents, we used different concentrations (25 µM and 250 µM) of the iron-chelating agent deferoxamine mesylate (D9533, Sigma-Aldrich, St. Louis, MO, USA). To assess the potential of bacteria to induce apoptosis in cancer cells, we used the apoptosis inducer Staurosporine (STS) (LC Laboratories, Woburn, MA, USA) at a concentration of 0.1 µM and the pan-caspase inhibitor Z-VAD-FMK (FMK001, R&D Systems, Minneapolis, MN, USA) at a concentration of 20 µM as control conditions.

MDA-MB-231 cells were cultured to a confluency of ca. 80% on either 6-well or 12-well plates, depending on the experiment. The cells were incubated in a humidified incubator at 5% CO₂ and 37 °C for 24–48 h. Subsequently, the media was changed to FBS supplemented DMEM without penicillin–streptomycin, and different concentrations of Magnetospirillum magneticum AMB-1 were added into the wells. Unless specified otherwise, the plates were always placed in a sealable box which was subsequently flushed with nitrogen for 15 min to produce hypoxic conditions. The plates were incubated using this setup at hypoxic conditions at 37 °C for 24–48 h. Using phenol red as pH indicator, the color of the media was monitored throughout the experiment, and a pH in the order of ~ 7.5 was ensured. To benchmark the iron-chelating potential of bacteria against known agents, we used different concentrations (25 µM and 250 µM) of the iron-chelating agent deferoxamine mesylate (D9533, Sigma-Aldrich, St. Louis, MO, USA). To assess the potential of bacteria to induce apoptosis in cancer cells, we used the apoptosis inducer Staurosporine (STS) (LC Laboratories, Woburn, MA, USA) at a concentration of 0.1 µM and the pan-caspase inhibitor Z-VAD-FMK (FMK001, R&D Systems, Minneapolis, MN, USA) at a concentration of 20 µM as control conditions.

C6 cells were plated in a 6-well cell culture plate (TPP 35 mm tissue culture plate, MidSci, St. Louis, MO, USA) and allowed to adhere overnight. The following day, cells were treated with the pan-caspase inhibitor zVAD-fmk (FMK001, R&D systems, Minneapolis, MN, USA) at a concentration 100 µM or with 100 µM of resveratrol. Two hours after pretreatment, cells were UV-irradiated (UV-C, 254 nm) for 5 min using a 3UV transilluminator (BioChemi System, UVP, Upland, CA, USA) as described by us previously [18 (link)]. 12 h after UV-irradiation, cells were harvested for immunoblot analysis, caspase activity measurements or stained for NFT formation using Thioflavin S.