Sc 514

Pyrrolidine dithiocarbamate (PDTC) and SC-514 (MedChemExpress, Monmouth Junction, NJ, USA) were used to inhibit NF-κB activation [21 (link), 22 (link)]. HCT-8 cells were pretreated with inhibitor for 2 h prior to C. parvum infection. PDTC and SC-514 were used at concentrations of 3.286 μg/ml and 22.43 μg/ml, which were not cytotoxic in either HCT-8 cells or C. parvum sporozoites.

We cultured A549 cell lines in McCoy’s 5A (GE Healthcare Life Sciences, Freiburg, Germany) plus 10% fetal calf serum (FCS; Thermo Fisher Scientific, Darmstadt, Germany) at 37°C. The medium contained penicillin and streptomycin. For A549 p53-Venus reporter cells [64 (link),65 (link)], selective antibiotics (400 μg/ml G418 (Carl Roth, Karlsruhe, Germany) and 50 μg/ml hygromycin (Thermo Fisher Scientific)) were added to maintain transgene expression.
For corresponding treatment of the cells, the medium was replaced with fresh one containing dimethyl sulfoxide (DMSO; Sigma-Aldrich) as a control, IKK2 inhibitor (15 μM TPCA-1, 120 μM sc-514 or 0.93 μM BMS-345541 (MedChemExpress, Sollentuna, Sweden)), 10 ng/ml or 50 ng/ml IL-6 (PeproTech, Hamburg, Germany) or 10 ng/ml TNFα (Enzo Life Science, Lörrach, Germany) 1 h before irradiating cells (if not stated otherwise) with X-rays at a dose rate of 1 Gy/26 s (250 keV, 10 mA).

Antibodies targeting the following proteins were used: TFRC (ab214039, Abcam, Cambridge, UK), FPN (NBP1-21502, Novus Biologicals, Englewood, CO, USA), FTH1 (ab65080, Abcam), SLC7A11 (600-401-GU3, Rockland Immunochemicals, Pottstown, PA, USA), GPX4 (ab125066, Abcam), GAPDH (sc-32233, Santa Cruz Biotechnology, Dallas, TX, USA), PGC1α (NBP1-04676, Novus Biologicals), SHARPIN (ABF128, Millipore, Burlington, MA, USA), β-actin (#4970, Cell Signaling Technology, Danvers, MA, USA), VDAC1/3 (ab14734, Abcam), PRMT5 (sc-376937, Santa Cruz Biotechnology), SDMA (SYM10; 07-412, Millipore), SOX10 (sc-365692, Santa Cruz Biotechnology), MITF (ab12039, Abcam), LC3B (NB100-2220, Novus Biologicals), NRF2 (#12721, Cell Signaling Technology), Parkin (#4211, Cell Signaling Technology), and BNIP3L/NIX (#12396, Cell Signaling Technology). For NRF2 detection, samples were pretreated with 50 μM MG132 (FUJIFILM Wako Pure Chemical Corporation, San Diego, CA, USA) for 3 h. Complex I/III/V was detected using Total OXPHOS Human WB Antibody Cocktail (ab110411, Abcam). Erastin, EPZ015666, ATN-161, and RGD peptide were purchased from Selleck Chemicals (Houston, TX, USA). TGF-β was purchased from BioLegend (San Diego, CA, USA). RSL3, SC-514, and ferrostatin-1 were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Deferoxamine was obtained from Cayman Chemicals (Ann Arbor, MI, USA).