Bipyridyl

3,3’-Diaminobenzidine (DAB)-enhanced Perls’ staining was used to detect iron accumulation in paraffin embedded lung sections according to the manufacture’s instructions. Briefly, sections of lung tissue were washed with PBS and incubated in fleshly prepared Perls’ solution (5% potassium ferrocyanide (Sigma-Aldrich)/10% hydrochloric acid) for 1 h, followed by a 15 min incubation in DAB. The labile iron pool of BEAS-2B cells was measured using Calcein AM method as follows. Briefly, 10⁶ BEAS-2B cells or HBEC were recovered using EDTA-free trypsin, counted and incubated in serum-free DMEM with 0.2 μM Calcein-AM at 37 °C for 7 min. Cells were subsequently washed and resuspended in Hanks’ Balanced Salts solution before the incubation with trypan blue (25 μg). Baseline fluorescence signal was measured at an excitation of 488 nm and emission of 517 nm. The increase in fluorescence upon 100 μM 2,2-Bipyridyl (Sigma-Aldrich, D216305) addition was recorded for each sample^{41 (link)}. Fe concentration in mouse lung homogenate is measured using inductively coupled plasma mass spectrometry (Agilent technology, 8900 ICP-QQQ) as previously described^{27 (link)}. Each mouse lung was normalized by protein concentration measured by BCA assay. Conditions for the ICP-QQQ were as follows: RF power was 1550 W. Sampling depth was 8.0 mm. Nebulizer flow rate gas was 1.05 l/min. Cell gas was 7.0 ml/min H₂. The isotope which measured was m/z 56. 0.05 mg/l of Co was added to each sample and was used as internal control.
Laser ablation of ICP-MS was analyzed to localize Fe in paraffin embedded mouse lung tissue using a solid state laser ablation system (Electro Scientific Industries, NWR 213). Conditions for the laser were as follows: 10 Hz repetition rate. Spot size was 25 μm. The scan speed was 25 μm/s and 0.8 l/min helium gas flow. Heat maps were generated using imaging software (iQuant2).
Total and ferrous iron in lung homogenates were analyzed by Iron assay kit (Abcam, 83366) according to manufacturer’s protocols.

Synthetic peptide Aβ(1-42): [H2N]-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA-[COOH] was purchased from Biopeptide. Preparation of the monomeric form of Aβ(1-42) was performed as described elsewhere37 (link). To do this, cold hexafluoroisopropanol (Fluka) was added to dry Aβ (1-42) to a concentration of 1 mM and incubated for 60 min at room temperature. Then this solution was put on ice for 10 min and aliquoted into non-siliconized microcentrifuge tubes (0.56 mg peptide per tube). Peptide in the tubes was dried under vacuum using Eppendorf Concentrator 5301. Dried peptide was stored at −80 °C. 2.5 mM peptide stock solution was prepared by adding 20 μL of 100% anhydrous DMSO (Sigma-Aldrich) to 0.22 mg peptide and incubating for 1 h at room temperature. For use in the experiments, the peptide was diluted to the required concentration with buffer solution. Equivalent amount of DMSO was added to the control samples in all experiments. Only freshly prepared peptide solutions were used for all experiments. By dynamic light scattering (DLS) and turbidity methods it was shown that there were no aggregates and higher molecular weight oligomers in Aβ(1-42) preparation (40 μM) after 1 hour following preparation of the water solution. To determine the turbidity the optical density at 405 nm of freshly prepared solutions of Aβ(1-42) was recorded on Jasco V-560 spectrophotometer within one hour. The absorbance of the solution does not change, which allows to make a conclusion about the absence of particles in solution with a size of 1–100 nm. DLS method allows measuring the particle size from 0.6 to 10 nm. The DLS measurements were carried out on a Zetasizer Nano ZS apparatus (Malvern Instruments Ltd.). According to our data the freshly prepared solution of amyloid (40 μM) does not contain particles in this size range.
The monomer and low molecular weight oligomer quantities in Aβ(1-42) solution were estimated by SDS-PAGE with pre-stabilization of oligomers by photoinduced crosslinking using covalent Tris (2,2-bipyridyl) dichlororuthenium (II) hexahydrate38 (link). The monomers constituted 80% in Aβ(1-42) preparation (Supplementary Figure S8).
Purified preparation of Na,K-ATPase (α1β1 isozyme) was obtained from duck salt glands as described elsewhere11 (link)39 (link). The purity grade of Na,K-ATPase was 99% (Supplementary Figure S9) and specific activity of the enzyme reached ~2400 μmol of Pi (mg of protein × h)⁻¹ at 37 °C.

The photoinitiator tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate (Ru(bpy)₃Cl₂·6H₂O) was prepared in the corresponding buffer to a concentration of 5 mM and stored at 4 °C. The electron acceptor ammonium persulfate (APS, chemical formula: (NH₄)₂S₂O₈) was prepared with a concentration of 2 M in the corresponding buffer, stored as aliquots at −20 °C, and thawed directly prior to usage.

GelMA constructs were prepared by adding 50% w/w of GelMA stock solution, 48% PBS, 1% of 50 mM tris(2,2-bipyridyl) dichlororuthenium(II) hexahydrate (Ru; Cat. No. 544981; Sigma-Aldrich, Auckland, New Zealand), and 1% of 500 mM sodium persulphate (SPS; Cat. No. 216232; Sigma-Aldrich, Auckland, New Zealand). Stock solutions of 50 mM Ru (MW = 748.63 g/mol) and 500 mM SPS (MW = 238.1 g/mol) were made in PBS. All solutions were sterilized with a 0.22 μm filter prior to use. Different gel consistencies (stiff, standard, and soft) were obtained by preparing different concentrations of the stock GelMA solution as per Table S2A in the Supplementary Materials. In the GelMA-AgNP constructs, the volume of AgNPs to be used was deduced from the total amount of PBS added. Constructs were pipetted into well plates and photo cross-linked for 3 min using visible light (Wavelength = 400–450 nm) at 30 mW/cm².

The estimation of the total ascorbate (AsA and DHA), reduced ascorbate (AsA), and dehydroascorbate (DHA) was carried out using the method of Gossett et al. [19 (link)]. In this method, the reduction of Fe³⁺ to Fe²⁺ with ascorbic acid in an acidic medium resulted in the formation of a red chelate between Fe²⁺, and 2,2′-bipyridyl was read at 525 nm. Dehydroascorbate was determined by subtracting AsA from AsA and DHA. The ascorbate content was calculated using a series of standards of L-ascorbate (Sigma Alrdich, MO, USA). GSH and glutathione disulfide (GSSG) were quantified by following the 5,5′-dithiobis (2-nitrobenzoic acid) method using glutathione reductase and 2-vinylpyridine and were measured at 415 nm according to the protocol of Griffith [20 (link)]. The GSH content was measured by subtracting the GSSG content from total glutathione content using the standard curve that was prepared with different concentrations of GSH.