Sineg

The ptfLC3 plasmid used here was a generous gift from Tamotsu Yoshimori (Addgene plasmid #21074; http://n2t.net/addgene:21074; accessed on: 30 January 2018; RRID: Addgene_21074, Watertown, MA, USA) [28 (link)]. Lipofectamine 2000 (ThermoFisher, Barcelona, Spain) was used to achieve transient transfection following the manufacturer’s instructions. The silencing of CHOP gene expression was performed by transient transfection of commercial siRNAs from Dharmacon (#J-004819-06, #J-004819-07, and #J-004819-08), which were introduced into the cells using RNAiMax (Life Technologies, Barcelona, Spain) during 24 h incubations. The DDIT3 sequence of CHOP was used to design the siRNA (accession number NM_004083 NCBI) and a negative siRNA control (siNeg) from Life Technologies was used as a control (unknown mixed sequences). The commercial inhibitors employed were: 3-methyladenine (3-MA, 5 mM; Merck KGaA, Darmstadt, Germany), Bafilomycin A1 (BafA1, 10 nM, Sigma), LY294002 (25 µM, Biogen Cientifica, Madrid, Spain), Oxythiamine chloride hydrochloride (OT, 1, 10 mM, or 100, 200, or 300 mg/kg mice; Santa Cruz Biotechnology, Heidelberg, Germany), and SP600125 (25 µM, Santa Cruz Biotechnology). The cells were seeded in culture plates and pre-treated with these commercial inhibitors for 1.5 h prior to their exposure to HCA for different times.

NARF2 and HT1080 cells were seeded in 6 well plates at a density of 2 × 10⁵ and 3 × 10⁵ respectively. Next day, the cells were transfected by 50 pmol of siRNAs against E2F1 or 50 pmol non-specific (scrambled/ siNeg) or Myc (10–50pmol) using Lipofectamine RNAi Max reagent according to the manufacturer’s instructions (Invitrogen life technologies). One day later, cells from multiple wells were expanded into 96 well plate for ferroptosis sensitivity assay or 6cm/ 9cm plates for western blot/ RNA extraction. siRNAs were obtained from Life technologies siE2F1 (cat#4427038), siMyc (cat#4427037) and siNeg (cat#4390843)

AS or BE cells (1 × 10⁶) were plated in 6-well plates and transfected for 72 h with Lipofectamine RNAiMax (Thermo Fisher Scientific) or siRNA directed to either control (siNeg), siSTRAP1, siSTRAP2, or a combination of the two STRAP siRNAs at 20 µM concentration with Lipofectamine RNAiMax (Thermo Fisher Scientific). siNeg (ON-TARGETplus Non-targeting siRNA #1) was obtained from Dharmacon (GE Dharmacon, Thermo Fisher Scientific, Lafayette, CO, USA). The siSTRAP1 (SASI_Hs01_00016957) and siSTRAP2 (SASI_Hs02_00343131) were obtained from Sigma Aldrich (St. Louis, MO, USA).

MDCK cells were seeded on 6-well plate in density of 70,000 cells and the next day were transfected with specific small interfering (si)RNA targeting α-catulin gene (siCTNNAL1) (HSS112822, Thermo Fisher Scientific) or with universal negative control siRNA (siNeg) (#12935200, Thermo Fisher Scientific) using Opti-MEM medium (Thermo Fisher Scientific, #11058021) and Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific, #13778100) according to manufacturer’s instructions. Before further experiments cells were cultured for 48 h. Effectiveness of silencing of α-catulin gene was confirmed by RT-qPCR. Independent MDCK cell lines were generated using lentiviral shRNA specific to canine α-catulin (KD1 and KD2) and non-effective shRNA scrambled cassette (control). Lentiviral cassettes were designed and cloned by OriGene (pGFP-C-shLentiviral vector, TR30023 and pGFP-C-shLenti-scrambled TR30021). Lentivirus was produced using Lenti-vpak Lentiviral Packaging Kit (Origene) according to the manufacturer protocol. GFP positive cells for stable cell lines were selected by FACS sorting.

Transfection of small interference RNAs (siRNAs) (Invitrogen), including MKRN1 siRNA (si-MKRN1: 5'-GCATGGAGGTGGTCTATGATT-3’) and negative control (si-Neg: Invitrogen #4390843), was implemented with the Lipofectamine RNAi MAX (Invitrogen). Cells were plated onto 12-well plates 24 h before siRNA transfection. Twenty-four hours after siRNA knockdown, cells were further transfected with Eag1 cDNA. Short hairpin RNAs (shRNAs) in the pLKO.1 puromycin-resistant vector (RNAi Core, Academia Sinica) include MKRN1-shRNAs (sh-MKRN1#1: TRCN0000289977, targeting human and rat MKRN1 sequences; sh-MKRN1#2: TRCN0000289976, targeting human MKRN1 sequence; sh-MKRN1#3: TRCN0000041206, targeting rat MKRN1 sequence) and GFP shRNA (sh-GFP: TRCN0000207020). For lentiviral packaging in HEK293T cells, virus-containing medium was harvested and concentrated using ultracentrifugation (10,000g for 4 h at 4 °C) to yield viral stocks. For lentiviral infection in HEK293T cells, viral stocks were supplemented with 8 μg/ml of polybrene (Sigma). Infected HEK293T cells were selected by 5 μg/ml of puromycin (Sigma) and subsequently transfected with Eag1 cDNA. For suppressing endogenous MKRN1 in cultured neurons, 7-day in vitro (DIV7) cortical neurons were infected with viral stocks supplemented with 0.4 μg/ml of polybrene. After puromycin selection, infected DIV11 neurons were subject to immunoblotting.