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The reaction was initiated by the addition of H2O2. A series of enzymatic reactions was activated by GSH-Px in the cells, which subsequently led to the conversion of GSH (reduced glutathione) to oxidized glutathione (GSSG). The change in absorbance Buparlisib during the conversion of GSH to GSSG was recorded spectrophotometrically at 412 nm. Statistical analysis Means from three independent experiments were reported. Two-way analysis of variance (ANOVA) and Student��s t-testing were applied for comparative analyses. A two-sided p-value of less than 0.05 was deemed significant. Results Successful transfection of HaCaT keratinocytes with adenoviral vectors A schematic of the experimental workflow is depicted in Figure 1. In order to further probe the relationship between p21 expression level and UVB irradiation on HaCaT keratinocytes, p21-overexpression (p21+), p21-interference (p21�C), and empty adenoviral vectors (CON) were transfected into HaCaT keratinocytes [10]. All adenoviral vectors were successful transfected and performed their function; as expected, the p21 mRNA and protein levels of p21+ cells were significantly upregulated compared to that of CON cells (p21+ vs. CON, pIPI-145 mw UVB exposure significantly decreases p21 mRNA expression across all ... Figure 3 p21 and p53 protein expression by western blotting. (A) Western blots of p21 and p53 expression with GAPDH as a reference standard. (B) p53 protein expression mirrored p21 protein expression. (C) As expected, p21 protein expression was significantly higher ... Effects of UVB irradiation on p21 expression in HaCaT keratinocytes To assess p21 expression after UVB radiation, HaCaT keratinocytes were treated with 0.2% serum for 24 h to reach quiescence (i.e., in actively-dividing cells, p21 is destabilized with a reduced 20�C60 min half-life [10,21]). UVB (30 mJ/cm2) was then applied to these HaCaT keratinocytes, which were harvested 1.5 h after Oxacillin exposure. Levels of p21 mRNA and protein expression were both found to be significantly downregulated after UVB exposure across all experimental groups (UVB-CON vs. CON, UVB-p21+ vs. p21+, UVB-p21�C vs. p21�C, p