Oligodt primer

Animals from each group were sacrificed (5–6 animals in each), and the brains were immediately dissected on ice. The hippocampus and the frontal–parietal cortex from the left hemisphere were used for protein extraction, while those from the right hemisphere were used for RNA extraction.
Total proteins were extracted using the Laemmli sample preparation protocol. Shortly, the tissue extracts were homogenized in RIPA buffer containing protease inhibitor cocktail (Sigma Aldrich, P8340) and left for incubation for 30 min at +4 °C. The samples were centrifuged at 15,000× g for 20 min. The supernatant was aspirated and transferred into a clean tube. Then, the supernatant was diluted with the Laemmli sample buffer (0.5 of the supernatant volume) and put to boil for 5 min. After that, the samples were put on ice and stored at −20 °C before the analysis.
Total RNA from rat neocortex and hippocampus was isolated using Extact RNA reagent (Evrogen, Moscow, Russia, BC032) according to the manufacturer’s instruction. The isolated RNA concentration was measured on a Shimadzu UV mini-1240 spectrophotometer. The RNA quality was assessed using agarose gel electrophoresis. RNA was reversely transcribed into cDNA using the MMLV RT kit (Evrogen, Moscow, Russia, SK022S) and oligo-dT primer (Evrogen, Moscow, Russia, SB001, PB006).

Total RNA was isolated using the Bio-Rad Aurum RNA mini-isolation kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. cDNA was synthesized by the Mint reverse transcriptase kit and oligodT primer (Evrogen). After that, qPCR was performed with ready to use SYBR Green HS mix (Evrogen) and the primers specific to the EGFR, PDGFRA, TNFA, BDNF, VEGFA, ITGA2, ITGA3, ITGV, HSP60, and KLF4 genes (Supplementary Table S1); coding EGFR; platelet-derived growth factor receptor alpha (PDGFRα); tumor necrosis factor-alpha (TNF-α); brain-derived neurotrophic factor (BDNF); vascular endothelial growth factor A (VEGFA); integrin subunits alpha 2, 3, 5, and V; heat shock protein 60 (HSP60); and Krüppel-like factor 4 (KLF4). Negative controls contained all the components of the PCR mixture with cDNA replaced by mRNA gave no signal. PCR reactions were carried out using the Roche LightCycler 96 amplifier (Roche, Basel Switzerland). The mRNA expression level was normalized to the S18 ribosomal RNA level for EVs composition analysis and to the S18 and RPL13a genes expression to study the EVs’ influence on the KLF4 expression using the LightCycler SW software (Roche).

Total cellular RNA was isolated using a single-step acidic phenol extraction as previously described^{4 (link)}. 1 µg total RNA was reverse transcribed with the 100U MMLV Reverse Transcriptase (Sibenzyme), 1 mM dNTP, 2 mM DTT, 2 µM OligodT primer (Evrogen) and standard thermocycler temperature conditions for MMLV. All real-time PCR reactions were performed using the ABI ViiA7 system (Thermo) and standard cycle. Amplifications were done using the real time PCR Master Mix qPCRmix-HS + LowROX (Evrogen) and primers and Taqman probes from the Supplementary Table 1.