Ultrapure water

DNA was extracted from young leaves (4 weeks post-germination) using a DNeasy plant kit (Qiagen). PCR validation of the existence of the newly discovered MITE family (Inbar) was performed using primers, designed using PRIMER3 version 4.0.0 (bioinfo.ut.ee/primer3/) from flanking sequences of the insertion. Each PCR reaction contained 12.5 μl ultrapure water (Biological Industries), 2 μl of 10xC Taq DNA polymerase buffer (EURX), 1.5 μl of 25 mM MgCl₂ (EURX), 0.8 μl of 2.5 mM dNTPs, 0.2 μl Taq DNA polymerase (5 U μl-1, EURX), 1 μl of each site-specific primer (50 ng/μl) and 1 μl of template genomic DNA (approximately 50 ng/μl). The PCR conditions were 94°C for 3 min, 30 cycles of 94° C for 1 min, 60°C for 1 min and 72°C for 1 min, and 72°C for 3 min. For sequence validation, PCR products were extracted from agarose gels using a QIAquick PCR Purification Kit (Qiagen) and subjected to sequencing.
Primer sequences can be found in S6 Table.

Self-assembled PEG-PCL micelles were prepared by surfactant-free nanoprecipitation with minor modifications of a previous protocol³² . PEG-PCL was dissolved in acetonitrile (ACN); ACN was then added dropwise into ultrapure water (Biological Industries, Kibbutz Beit-HaEmek, Israel) at a ratio of 1:2 (v/v), and stirred until complete solvent evaporation of ACN was obtained. The CMC of the polymer was determined by enhancement of the absorbance of the hydrophobic dye DPH upon entrapment within micelles. The PEG-PCL solution was prepared at various concentrations. Then, DPH (dissolved in ethanol) was added at a final concentration of 4 μM. The relative absorbance at 378 nm/400 nm was plotted against the PEG-PCL concentration, and the point at which the two lines intersect was defined as the CMC of the copolymer^{56 (link)}.

Peptide segments ¹⁵⁶NTVTFN¹⁶¹, ¹⁶⁸SIAVNF¹⁷³, ¹⁹⁶IATLYV²⁰¹, ³²⁴GIVIVA³²⁹ and ³⁶⁹TSYVGV³⁷⁴ from Als5 (UniProt accession number Q5A8T7) were synthesized at >98% purity and purchased from GL Biochem (Shanghai) Ltd. The peptides were synthesized with unmodified termini for crystallography or with fully capped termini (acetylated in the N-terminus and amidated in the C structural terminus) for fibrillation assays. Thioflavin T (ThT) was purchased from Sigma-Aldrich. Dimethyl sulfoxide (DMSO) was purchased from Merck. Ultra-pure water was purchased from Biological Industries.

Activated carbon powder (YP-50F, Kuraray Chemical Co. Ltd., Osaka, Japan) was used as the electrode active material. Ammonium chloride (NH₄Cl, Daejung Chemicals & Metals Co., Siheung, Republic of Korea) was used for doping the activated carbon powder with nitrogen. We specifically selected ammonium chloride that were an alkaline substance with high solubility in water, as compared to urea, glycine, nitric acid, etc., to minimize the effect of precursor type and concentration on the LPP process in terms of both conductivity and pH. However, a significant reduction of pH was still observed in the reactant solution after the LPP reaction. Nitrogen-doped activated carbon powder was subjected to the LPP reaction to impregnate iron oxide nanoparticles using iron chloride tetrahydrate (FeCl₂·4H₂O, Kanto Chemical, Tokyo, Japan) as the precursor. Cetrimonium bromide (CTAB, CH₃(CH₂)₁₅N(Br)(CH₃)₃, Sigma-Aldrich, St. Louis, MO, USA) was added to the LPP reactant solution to disperse iron oxide nanoparticles on the surface of nitrogen doped activated carbon. All chemicals used in this study were reagent-grade chemicals and ultra-pure water (Biological industries, Beit, Israel) was used to prepare the aqueous LPP reaction solutions.

Bovine serum albumin (BSA) and palmitate conjugated to BSA were prepared according to a previously described protocol (52 (link), 54 (link), 55 (link)). Briefly, to prepare the BSA solution, 20% (w/v) fatty acid free bovine albumin (MP Biomedicals, Cat #0219477205) was gradually added to Ultra Pure water (Biological Industries, Cat #01-866-1A) at 52°C with gentle agitation until the BSA was fully dissolved, yielding a transparent yellow solution. To prepare a palmitate-BSA solution, 2.6% (w/v) of palmitic acid (Sigma-Aldrich, Cat #P0500) was added to 0.1 M NaOH (Sigma-Aldrich, Cat #221465) and heated to 70°C until fully dissolved, yielding a concentration of 100 mM. To conjugate palmitate to BSA, the 100 mM stock of palmitate in 0.1 M NaOH was added to 20% BSA at a ratio of 1:9 and heated at 55°C for 10 min. After conjugation, both the 20% (w/v) BSA solution and 10 mM Palmitate-BSA solutions were filter sterilized using a 0.45 μm PES filter (Celltreat, Cat #229709). Solutions were then aliquoted and stored at −20°C. Before use, solutions were warmed to 37°C. The total amount of BSA was held constant in all conditions by mixing ratios of fatty acid free BSA to palmitate-BSA as follows: BSA only (4:0), 0.1 mM palmitate-BSA (3:1), 0.2 mM palmitate-BSA (2:2), and 0.4 mM palmitate-BSA (0:4).

Polymetric NPs containing both JO and PTX (NPs/PTX-JO) and NPs lacking JO (NPs/PTX) were prepared by (low MW)-surfactant-free nanoprecipitation [28 (link)]. Different amounts of JO (1.5, 4.8, 9.6, and 19.2 μL) were dissolved in 0.8 mL acetonitrile (ACN) containing 70 μM PP and 14 μM PTX. Thereafter, ACN was added dropwise into ultrapure water (Biological Industries, Kibbutz Beit-HaEmek, Israel), and stirred at room temperature to obtain NPs with different PTX:JO molar ratios of 1:0.16 (1:1 w/w [29 (link)]),1:0.5, 1:1, and 1:2, respectively. S15-APTs were covalently conjugated to the NPs as previously described [14 (link)].

Primers for a subset of insert sites were designed in Primer3 software (See primer sequences in Table S3, http://bioinfo.ut.ee/primer3-0.4.0/) for PCR validation. Each PCR reaction was prepared with 10 μl of PCRBIO HS Taq Mix Red (PCRBiosystems), 7 μl of ultrapure water (Biological Industries), 1 μl of the site-specific primer (10 μM) and 1 μl of template genomic DNA (~ 50 ng/μl). The PCR conditions were 95°C incubation for 2 min, 40 cycles of 95°C for 10 s, the specific annealing temperature (calculated according to each primer set) for 15 s and 72°C for 15 s. PCR products were tested on 1.5% agarose gels and visualized with ethidium bromide (Amresco). Expected product sizes were determined by a DNA size standard (100 bp ladder, SMOBIO), and for some insertions the amplified products were extracted from gel and sequenced for validation.