Comparative Analysis of AGE and RAGE Levels in Human Somatic and Embryonic Stem Cells under H2O2-Induced Noncytotoxic Oxidative Stress Conditions
Hydrogen peroxide treatment diagram, dose-response curves following hydrogen peroxide (H2O2) 72 h exposure and relative amount of CML in protein extracts of hES and somatic cells after 72 h of noncytotoxic H2O2 oxidative stress treatment. (a) Hydrogen peroxide treatment diagram. Representative scheme for 72 h H2O2 oxidative stress treatment. At the end of the protocol, 3 days after the initiation of chemical exposure, the viability, gene expression, and protein levels were evaluated in exposed versus not exposed control (CTR) cells. (b) Dose-response curves, modified from Barandalla et al. . HUES3, HUES7, Hs27, and HUVEC were exposed to increasing concentrations of hydrogen peroxide for 72 hours, and cell viability was determined by AlamarBlue reagent. Blue dotted square highlights noncytotoxic range for HUES3, HUES7, and Hs27 cells, between 4 and 16 μM. Orange solid square highlights noncytotoxic range for HUVEC, between 4 and 32 μM. Data (means ± SD, 3 separate replicates per H2O2 experimental condition) are expressed as percentages of cell viability relative to the respective CTR, untreated control cells. (c–f) Relative amount of CML in protein extracts of hES and somatic cells after 72 h of noncytotoxic H2O2 oxidative stress treatment. Relative amount of protein-bound Nε-carboxymethyl-lysine (CML) in the H2O2-treated cells normalized with the nontreated cells. The CML amount is related to the protein load (Ponceau S staining). A representative slot blot is shown for each cell line. PBS was used as a negative control (arrows). The quantification was performed by slot blot analysis: mean ± SD, 3 separate replicates per H2O2 experimental condition. Statistically significant differences between groups are indicated as follows: and as established by one-way ANOVA and Tukey’s post hoc test.
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