Porcine hemin

Human erythroleukemia K562 cells were used in this study (not an authenticated cell line; available at our institute; periodically tested for mycoplasma). Cells were grown at 37 °C at 5% CO₂ in RPMI 1640 (Gibco) with 10% FBS (Sigma) and 1% penicillin–streptomycin (Gibco). Density was kept between 1 × 10⁵ and 5 × 10⁵ cells per ml medium. Cells were routinely tested for mycoplasma. Prior to every experiment, cells were weekly bulk-sorted for 2 consecutive weeks on Becton Dickinson SORP FACSAria FUSION Flow Cytometer. Gating was based on cells without an enhancer (no-E cells) or fluorescence-minus-one controls. Flow cytometry analysis was done 3 days after sorting on a Beckman Coulter Cytoflex S. For differentiation, culture medium was supplemented with porcine hemin (Sigma, 30 µM final concentration) 1 day after sorting. Hemin and medium were refreshed 1 day later. We prepared 4 mM hemin stock solutions according to ref. ^{62 (link)} and kept them at −20 °C. For long-term culturing and FI monitoring, at each indicated time point an aliquot of cells was taken and treated with hemin for 2 days, and FI was monitored. To obtain irreversibly silenced populations of cells, GFP-negative cells were weekly sorted for 6 (E100) or 9 (E407) consecutive weeks.

All bacterial strains generated and/or used in this study are listed in Key resources table. For growth in iron limited conditions, bacteria were grown overnight in Tryptic Soy Broth (TSB) (Oxoid). Cells were harvested by centrifugation, washed with RPMI containing 10 µM EDDHA (LGC standards), adjusted to an OD₆₀₀ = 1 and 2,5 µl were used to inoculate 0,5 ml of RPMI+ 1% casamino acids (BACTO) + 10 µM EDDHA in individual wells of a 48 well microtiter plate (NUNC). As sole iron source 200 nM porcine hemin (Sigma), 2.5 µg/ml human hemoglobin (own preparation), 10 µg/ml human myoglobin (Sigma) or equine myoglobin (Sigma), 117 nM human haptoglobin-hemoglobin or 200 nM hemopexin-heme (Sigma) were added to the wells. Bacterial growth was monitored using an Epoch2 reader (300 rpm, 37°C). The OD₆₀₀ was measured every 15 min.

A sample of 5 mg of β-hematin (lot HMZ-38-01) was purchased from InvivoGen (Toulouse, France). This sample is referred to as “commercial β-hematin” throughout the paper.
A second sample of β-hematin was synthesized according to published protocols^{29 (link),30 (link)} with slight modifications; briefly, a solution of 4.54 mM porcine hemin (Sigma-Aldrich, >98% pure) in 0.04 M NaOH was adjusted to pH 4.0 with 2% propionic acid dropwise and incubated at 70°C for 18 h. The formed crystals were filtered on cellulose, washed with 1 M acetic acid, dried over phosphorus pentoxide, manually powdered and stored at 4°C. The powder was characterized by infrared spectroscopy, yielding 3 bands characteristic of hemozoin^{7 (link)} at 1711, 1662 and 1209 cm⁻¹. This sample is referred to as “Mons β-hematin” throughout the paper.