Following incubation, the cells were lysed with lysis buffer supplied by the assay manufacturer. of application: anticancer therapy, scintigraphy, and cosmetology. The experiments were performed on immortalized human umbilical vein endothelial cells (HUVEC-STs). Light contrast phase PHA-793887 microscopy as well as cell viability assays showed that only Pluronic F127 MICELLES decreased the number of HUVEC-STs in contrast to PLA/MMT/TRASTUZUMAB, PLA/EDTMP, and PLGA/MDP NPs, which altered cell morphology, but not their confluency. The tested NPs induced not only DNA strand-breaks and alkali-labile sites, but also internucleosomal PHA-793887 DNA fragmentation, visualized as a DNA ladder pattern typical of apoptosis. Moreover, generation of free radicals and subsequent mitochondrial membrane potential collapse showed the significance of free radical production during interactions between NPs and endothelial cells. High concentrations of NPs had different degrees of toxicity in human endothelial cells and affected cell proliferation, redox homeostasis, and triggered mitochondrial dysfunction. software70. RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) ATM, ATR, AIF, Bax, Bcl-2, Casp-3, H2AX, and PARP (the primer sequences are listed in Table ?Table2)2) mRNA expression levels were measured by qRT-PCR as described previously71. Total RNA was extracted using the TRI Reagent (Sigma-Aldrich, USA) according to the manufacturer instructions, and reverse transcription to cDNA was carried out using the TaqMan Reverse Transcription Reagents (Thermo Scientific, USA). The qRT-PCR was performed with the SYBR-green PCR master mix (EURx, Gdansk, Poland) in an Eco Real-Time PCR System (Illumina, USA). The cycling conditions were 94?C for 4?min, followed by 40 cycles at 94?C for 15?s, 60?C for 25?s, and 72?C for 25?s. The housekeeping gene hypoxanthine phosphoribosyltransferase 1 (HPRT1) served as an internal control and Eco Real-Time PCR Relative Quantification Software (Illumina, USA) was ART4 used for quantification. Data (Ct values) were transformed into relative copy number values (the number of mRNA copies of the examined genes per housekeeping gene index), calculated as the average Ct value of the HPRT1 housekeeping gene, and standardized to the level of mRNA transcripts in untreated cells, taken as 1. Table 2 Primer sequences used for RT-PCR. PHA-793887 for 10?min and the supernatant containing low molecular weight DNA (LMW DNA) was treated with RNase A (1?mg/mL) and Proteinase K (1?mg/mL) for 30?min at 37?C. DNA samples were fractioned by electrophoresis on 1.8% agarose gel stained with ethidium bromide (0.5?mg/mL) for 2?h at 100?V in TBE buffer (100?mM Tris HCl, 0.1?M boric acid, and 1.5?mM EDTA, pH 8.0). The gels were visualized under UV illumination using an In Genius Bio Imaging System (Syngene International Limited, India) in the Department of Molecular Genetics, University of Lodz. HISTONE H2AX phosphorylation HISTONE H2AX phosphorylation was measured using the Human Phospho-Histone H2AX DuoSet IC ELISA kit according to the manufacturer instructions (R&D Systems, UK). A total of 2??106 cells were incubated for 24, 48, and 72?h with NPs. Following incubation, the cells were lysed with lysis buffer supplied by the assay manufacturer. Clarified cell extracts were added to triplicate wells to determine the cellular level of phospho-histone H2AX and total histone concentration. The resulting fluorescence was measured on a Fluoroskan Ascent plate reader (Fluoroskan Ascent FL, Sweden) with filter pairs of 540/600?nm and 360/450?nm. The results were presented as a fluorescence ratio measured at 540/600?nm to that measured at 360/450?nm (phosphorylated form to total Histone H2AX concentration) relative to the control cells. Apoptosis and necrosis detection The number of cells in various stages of cell death were analysed by double staining with Hoechst 33258 and PI as described previously72. Cells were cultured with various concentrations of NPs (100?g/mL of PLA/MMT/TRA, PLA/EDTMP, PLGA/MDP, and 0.025?g/mL of Pluronic F127 Ms) for 24, 48, and 72?h. At certain time points, the cells were removed from culture dishes by trypsinization, centrifuged, suspended in HBSS at a final concentration of 1 1??106 cells/mL, and then.

Following incubation, the cells were lysed with lysis buffer supplied by the assay manufacturer