Nanodrop one spectrometer

The total RNA of the target sample was extracted using the Trizol RNA isolater Total RNA Extraction Reagent (Vazyme Biotech, Nanjing, China) following the manufacturer’s protocol. The quality and concentration of the isolated RNAs were assessed using a NanoDrop One Spectrometer (Thermo Fisher Scientific, USA). Next, 400 ng of each RNA was reverse transcripted into cDNA using the HiScript II Q RT SuperMix (+ gDNA wiper) kit (Vazyme Biotech, Nanjing, China) in line with the protocol. Finally, qRT-PCR was carried out with an ABI QuantStudio 5 equipment (Thermo Fisher Scientific, USA) in a 10-µL reaction system, containing 5µL of ChamQ SYBR Color qPCR Master Mix (Vazyme Biotech, Nanjing, China), 2.4µL of ddH₂O, 2µL of 20-fold diluted cDNA, and 0.3µL of each 10 µmol primer. The procedure of qPCR comprised an initial denaturation step at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 10 s, and primer annealing together with amplification at 60 °C for 30 s. With 2^−ΔΔCt (Ct: cycle threshold) method [58 (link)], the relative expression level of the target gene was calculated, with N. lugens 18 S ribosomal RNA (Nl18S, GenBank accession number: JN662398.1) serving as the housekeeping gene. Primers used for qRT-PCR are listed in (Supplementary Table 4 A).

Aβ_1-42 monomers were prepared as described previously (Sinha et al., 2011 (link)). The lyophilized Aβ_1-42 peptides were dissolved in HFIP at a concentration of 1 mg/ml and sonicated for 10 min in an ice-bath, followed by incubation with shaking at 4°C for 3 h. Subsequently, the resulting solution was evaporated under a gentle stream of N₂ gas to remove HFIP, and peptide films were formed. Thereafter, the peptide films were dissolved in 10 mM sodium hydroxide alone with sonication for 1 min and centrifuged at 4°C (16000 × g, 10 min). Finally, the supernatant was collected and quantified using a NanoDrop One spectrometer (Thermo Fisher Scientific, Waltham, MA, United States) at 280 nm using an extinction coefficient of 1490 M⁻¹ cm⁻¹.
To prepare different aggregation stages of Aβ_1-42, Aβ_1-42 monomers were diluted in phosphate-buffered solution (PBS, 10 mM, pH 7.4) at a final peptide concentration of 50 μM and incubated with continuous orbital shaking at 150 rpm. At different incubation time points, Aβ_1-42 solution was collected for corresponding experiments.

Total RNA from the early and late passages were extracted with a Zymo Quick-RNA MiniPrep kit (R1054, Zymo Research, Tustin, CA, USA) according to the manufacturer’s instructions. The RNA concentration and purity were evaluated with a NanoDrop One spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The SensiFAST cDNA synthesis kit (BIO-65053, SensiFAST, Bioline, Memphis, TN, USA) was used to synthesize cDNA from RNA. Each PCR reaction was conducted in a 20 μL reaction mixture with a final amount of 10 ng cDNA for each mixture using the SensiFAST SYBR No-ROX qPCR kit (BIO-98005, Bioline, Memphis, TN, USA). Quantitative RT-qPCR experiments were carried out with the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, Berkeley, CA, USA). The cycling parameters were as follows: 95 °C for 2 min, followed by 40 cycles at 95 °C for 5 s, an optimized annealing temperature for 10 s, and 72 °C for 5 s. The results of each target gene were normalized to β-actin mRNA expressions. The primer sets were designed using the Primer3 online software (https://primer3.ut.ee/) (accessed on 21 November 2021). The list of primers used for the experiments is shown in Table S1. Each experiment had three replicates per condition, and a statistical analysis was performed with a Mann–Whitney t-test using GraphPad Prism 9.3.1.