Figure 5: Proteasome inactivation following plasma exposure.
(A) HaCaT, HCT-116 and SK-MEL-28 cells were exposed to plasma treatment (He, He-O2 and He-N2) for 5 min with 1 hr post-treatment storage. Proteasome chymotrypsin-like activity was measured 24 hr post treatment using the fluorogenic peptide LLVY-AMC. Proteasome activity is presented as a percent of non-treated cells. Data, mean ± SEM from three independent cultures, *P < 0.05; **P < 0.01. (B) Effect of plasma activated liquid (PAL) on cell proteasome activity. HaCaT, HCT-116 and SK-MEL-28 cells were exposed to PAL for 1 hr (PBS treated for 5 min with He plasma (580 μM H2O2 and 300 μM NO2− measured in the PAL); He-O2 plasma (40 μM H2O2 and 50 μM NO2− measured in the PAL) and He-N2 plasma (390 μM H2O2 and 300 μM NO2− measured in the PAL) or to a mix of 580 μM hydrogen peroxide and 300 μM mM NO2− for HaCAT cells and proteasome activity was measured 24 hr post treatment. Data, mean ± SEM from three independent cultures, *P < 0.05; **P < 0.01. (C) Effect of glutathione peroxidase overexpression on apoptosis following plasma treatment. HaCaT cells were grown in medium depleted or supplemented in Selenium (Se), a condition which is known to increase glutathione peroxidase activity and were exposed to He-plasma treatment for 5 min with 1 hr post-treatment storage. Cells were stained with Annexin V-FITC and PI and analyzed by flow cytometry 24 hr after plasma treatment. Percentage of apoptotic cells (Annexin-PI positive) was shown by histogram. (D) Effect of glutathione peroxidase activation on proteasome activity following plasma treatment. HaCaT cells were grown in medium depleted or supplemented in Selenium (Se), a condition which is known to increase glutathione peroxidase activity and expression and then exposed to CAPP treatment (He, He-O2 and He-N2) for 5 min with 1 hr post-treatment storage. Proteasome activity was measured 24 hr post treatment. Data, mean ± SEM from three independent cultures, *P < 0.05; **P < 0.01.
