Ferroptosis-Induced mRNA Abundance Changes

To assess changes in mRNA abundance following induction of ferroptosis, cells were seeded in a 6-well plate at 1 × 10⁵ cells/well and incubated for 24 h before co-treatment with 10 µM erastin and 10 µg tetracycline for 24 h. Total RNA was isolated using TRIzol reagent (ThermoFisher; Waltham, MA, USA). RNA purity and integrity were confirmed by Nanodrop (ThermoFisher) and agarose gel electrophoresis, respectively, before reverse-transcription using SuperScript II (Invitrogen, Carlsbad, CA, USA). The relative abundance of TFRC, NCOA4, ATG5, CISD1, SLC7A11, and ferroportin (Supplementary Figure S3) were determined by real-time qPCR using SYBR green chemistry on an ABI 7900HT Real-Time PCR system (ThermoFisher; Waltham, MA, USA) and normalized relative to peptidylprolyl isomerase B (PPIB) abundance using the 2^−ΔΔCt method (User Bulletin no. 2, Applied Biosystems). Primer sequences for each mRNA of interest were obtained from previously published sources: PPIB [22 (link)], TFRC [44 (link)], NCOA4 [45 (link)], ATG5 [46 (link)], CISD1 [47 (link)], and SLC7A11 [48 (link)].