The influence of the water-soluble  fullerene derivative containing five residues of 3-phenylpropionic acid and a chlorine addend appended to the carbon cage (F-828) on serum-starving human embryo lung diploid fibroblasts (HELFs) was studied. responses to intrinsic and extrinsic cellular stresses . Fullerene derivative C60(C(COOH)2)3 (C60-COOH) was investigated previously and it was demonstrated that C60-COOH pretreatment attenuated the lipopolysaccharide-mediated activation of nuclear factor- (NF-) cytokine while the NRF2 activity decreases . Thus serum-starving HELFs represent a good model to study water-soluble fullerene-mediated NRF2 induction and NF-(F: GCCTTCTTTGAGTTCGGTGG R: ATCTCCCGGTTGACGCTCT);? (F: TACAGGCTGGCTCAGGACTAT R: CGCAACATTTTGTAGCACTCTG);? (F: CGACGAGTTTGAACTGCGGTA R: GGGATGTCAGGTCACTGAATG);? (F: GAATCTGGTTTCAGCTAGTCTGG R: GGTGGGAGATAATGAATGTGCAA);? (F: AAGCTACCTCTCAGCCTACTTT R: CCACTGTTTTCTGTACCCGGA);? (F: GTGGTGTCCATTGAGGGTATCC R: GCTCAGCGAAGTTGGCGAT);? SLx-2119 (F: CAGATGGCCCATACCTTCAAAT R: CGGAAACGAAATCCTCTCTGTT);? (F: SLx-2119 TCCAGTCAGAAACCAGTGGAT R: GAATGTCTGCGCCAAAAGCTG);? (F: TTGGGGCTAGGATTGTGTCTA; R: GAGTGTTCGGCACATGGGTA);? (F: CCCGAGAGGTCTTTTTCCGAG R: CCAGCCCATGATGGTTCTGAT);? (reference gene) (F: GCCCGAAACGCCGAATAT R: CCGTGGTTCGTGGCTCTCT). Standard curve method was used for the quantification of RNA levels. 2.6 Statistics All the reported results were reproduced at least three times as independent biological replicates. In flow cytometry the median of signal intensities was analyzed. The figures show the mean and standard deviation (SD) values. The significance of the observed differences was analyzed with nonparametric Mann-Whitney tests. values < 0.05 were considered statistically significant and marked in figures with (< 0.01) and became higher than that of the control experiment when similarly cultured serum-free cells had been exposed to F-828 (0.2-0.5?< 0.01). The ratio of the cells in the G0/G1 cycle phase reduces (< 0.05). Propidium iodide staining for DNA content material has exposed that HELF inhabitants expanded in serum-free press shows an elevated contribution through the G2/M cells (23% versus 7% for the moderate with 2% FBS) Shape 4(a). An publicity from the SLx-2119 cells to 0.1-0.25?BAXgene mixed up in apoptosis induction. It had been revealed how the known degree of theBAXmRNA increased in the current presence of F-828. F-828 resulted in decreased expression from the antiapoptotic genesBCL2andBCL2A1 BCL2L1BIRC2BIRC3viaradical addition pathway. Additionally it is possible how the fullerene derivative affects the enzymes and transcription elements in charge of ROS creation and removal in the cell. 3.7 F-828 Causes a Reduction in the amount of NOX4 Protein in Serum-Starving HELFs It's been demonstrated that creation of cellular ROS relates to the action of NAD(P)H-oxidase kind of enzymes predominantly those SLx-2119 encoded by NOX gene family SLx-2119 members . NAD(P)H-oxidase 4 (NOX4) continues to be recognized recently as a major source of ROS in HELFs and it was shown to be implicated in the fibrogenic response to lung injury . In living cells NOX4 catalyzes the reaction responsible for the hydrogen peroxide formation. The level of NOX4 protein was decided in HELFs using FCA and antibodies SLx-2119 specific to NOX4 (Physique 7). The population of serum-starving HELFs comprises two cell fractions: one with elevated NOX4 (gate R around the plot of FL1-NOX4 versus SSC) representing about 60% of the total amount of the cells and the other with a lower NOX4 content. For comparison HELFs cultivated in the presence of 2% FBS contain just 7% of cells with high level of NOX4 protein. Physique 7 F-828 entails a decrease in the level of NOX4 protein in serum-starving HELFs. (a) (FCA): (1): the FL1-NOX4 versus SSC plots. Gate R encircles the fraction of HELFs with elevated Ets2 values of FL1-NOX4; (2): dependence of the median values of the FL1-NOX4 … The mean level of NOX4 protein in HELFs cultivated under the serum starvation conditions is 3 times higher than that in the cells grown in the medium made up of 2% of FBS (Physique 7(a)). Interestingly the rate of DCF production in the serum-starved cells also appeared to be 3 times higher than in the control cells which were cultivated in the presence of 2% of serum (Physique 6). The.