Ds 11 spectrophotometer

Triple coculture cells were lysed with LBP lysis buffer (RNA extraction kit, NucleoSpin RNA Plus, Macherey–Nagel, Düren, Germany) and homogenized by transferring the lysates to QIAShredder membranes (Qiagen, Hilden, Germany), followed by centrifugation for 2 min at 20 913 × g (Eppendorf 5804 R, Hamburg, Germany). Lysates were stored at -80 °C. RNA was further extracted with the NucleoSpin RNA Plus extraction kit (Macherey–Nagel, Düren, Germany). RNA concentrations were determined with a Denovix DS-11 spectrophotometer (Denovix, Wilmington, USA). Next, 1 µg RNA was treated with DNase I (1 U/µL, Thermo Fisher Scientific, Merelbeke, Belgium) in a total reaction volume of 25 µL, diluted with H₂O (UltraPure DEPC-Treated Water, Thermo Fisher Scientific) for 30 min at 37 °C. The enzymatic reaction was stopped with 1 µL EDTA (50 mM) and an incubation of 10 min at 65 °C. A260/A280 ratios of all samples were between 1.8 and 2.1 (Denovix DS-11 spectrophotometer).

Total RNA from HuR-NP- and C-NP-treated cells and untreated control cells were isolated using Trizol reagent (Life Technologies, Grand Island, NY, USA), and the RNA quality was determined using Denovix DS11 spectrophotometer. Complementary DNA (cDNA) was synthesized from 1 µg RNA/sample using a QuantScript cDNA synthesis kit (Bio-Rad, Richmond, CA, USA). An amount of 3 µg of synthesized cDNA, quantified using Denovix DS11 spectrophotometer, was subjected to real-time quantitative reverse transcriptase (qRT)-PCR (Bio-Rad CFX96™TouchReal-Time PCR Detection System; Richmond, CA, USA) using the premix iQ SYBR green qRT-PCR kit (Bio-Rad) as previously described [30 (link),41 (link)]. The oligonucleotide primers (Integrated DNA Technology, Coralville, IA, USA) and their sequences for the amplification of HuR, BCL-2, and 18S RNA are shown below.
The PCR cycling parameters and all other experimental conditions followed have previously been described [41 (link)]. The cycle threshold (Ct) value assessed by qRT-PCR was calculated for the transcripts and was normalized to a housekeeping gene. The changes in mRNA expression levels were expressed as fold change relative to control. Each sample was run in triplicate. The experiments were repeated at least three times for reproducibility and subjected to statistical analysis.

FL WT tau monomer was filtered through a 100 kDa molecular weight cut-off filter (Corning) as instructed by the manufacturer (15,000g for 15 min). Filtered protein was collected, and protein concentration determined via DeNovix DS-11 Spectrophotometer. RNA or DNA was boiled for 7 min and snap-cooled on ice before addition to filtered monomer at determined mass ratio. All nucleic acid concentrations were quantified on a DeNovix DS-11 Spectrophotometer. Tau monomer was brought to a final concentration of 8 μM using tau buffer (10 mM Hepes, 100 mM NaCl in Millipore H₂O). DTT was added at a final concentration of 11% before adding inducer (RNA, DNA, heparin) or tau buffer. Fibril mixtures were agitated at 350 rpm using a Thermomixer (Eppendorf) set to 37 °C for determined time points.