Volume 112, Issue 11, Pages 2397-2407 (June 2017) Role of Ambient Gas Composition on Cold Physical Plasma-Elicited Cell Signaling in Keratinocytes  Anke Schmidt, Sander Bekeschus, Helena Jablonowski, Annemarie Barton, Klaus-Dieter Weltmann, Kristian Wende  Biophysical Journal  Volume 112, Issue 11, Pages 2397-2407 (June 2017) DOI: 10.1016/j.bpj.2017.04.030 Copyright © 2017 Biophysical Society Terms and Conditions

Figure 1 Impact of shielding gas on cell viability in human keratinocytes. Scheme (left) and image (right) of the kINPen 09 plasma jet equipped with a shielding gas device. A 60-mm petri dish containing 5 mL Roswell Park Memorial Institute 1640 medium was plasma treated for 180 s, and the liquid was immediately added to HaCaT cells for 3 h (a). Afterwards, the cell culture medium was replaced. The shielding gas mixture during plasma treatment was varied in five steps, ranging from pure nitrogen to pure oxygen. The x axis indicates the O2 amount of the O2 to N2 shielding gas mixture in percentage. Quantitative analysis of percentages of dead cells (mean fluorescence intensity) shows either cytotoxicity measured by a CellTox Green assay (b) or caspase 3 activity analyzed by a Green Caspase 3 Staining Kit (c) after 24 h. Un- or H2O2-treated cells (100 μM) were used as controls. Data are mean ± SD of three independent experimental repetitions and technical triplicates. Asterisk (∗) presents a significant difference (α = 0.05) measured using Tukey’s multiple comparisons posttest in one-way ANOVA. Biophysical Journal 2017 112, 2397-2407DOI: (10.1016/j.bpj.2017.04.030) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 2 Shielding gas composition affects gene expression signature of HaCaT cells detected by microarray. HaCaT cells were exposed to plasma-treated medium (either for 20 or 180 s) for 3 h, and microarray analysis was conducted. Heat map illustrates the fold regulation of genes, which were significantly upregulated (plus twofold; blue), downregulated (minus twofold; red), or not regulated (white) by cold plasma. The amount of O2 was given in percent. The numbers represent significantly changed genes in comparison to untreated cells (a). Fold regulation of plasma-treated transcripts were detected by microarray, which were found to be differentially expressed in all groups (mean level) (b). Three independent experimental repetitions and technical triplicates were performed. Biophysical Journal 2017 112, 2397-2407DOI: (10.1016/j.bpj.2017.04.030) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 3 Plasma shielding gas variations induce differential gene expression signatures in HaCaT keratinocytes. Quantitative gene expression levels of VEGFA (a), HBEGF (b), CSF2 (c), PTGS2 (d), and IL-6 (e) 3 h after exposure to plasma-treated medium (180 s) are depicted. Values in the area between lines were not significantly changed (plus twofold upregulated; downregulated below 0.5). Analysis was done with Tukey’s multiple comparisons test and one-way ANOVA with a significance level of α = 0.001 (∗∗∗). Bars and error bars are mean ± SD. Biophysical Journal 2017 112, 2397-2407DOI: (10.1016/j.bpj.2017.04.030) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 4 Extracellular concentration of cytokine and chemokine signaling proteins. Secretion of VEGFA 24 h (a); GM-CSF 6 h (b); IL-6 18 h (c), and IL-8 18 h (d). Keratinocytes were incubated with plasma-treated medium (using a shielding device and varying concentrations of O2 and N2) or H2O2 (100 μM). Analysis was done with Tukey’s multiple comparisons test and one-way ANOVA with a significance level of α = 0.05 (∗), α = 0.01 (∗∗), and α = 0.001 (∗∗∗). Four experimental repetitions and technical triplicates were measured. Bars and error bars are mean ± SD. Biophysical Journal 2017 112, 2397-2407DOI: (10.1016/j.bpj.2017.04.030) Copyright © 2017 Biophysical Society Terms and Conditions

Figure 5 Shown here is the concentration of reactive oxygen and nitrogen species in plasma-treated liquids (a) and the proposed signaling pathways in this study (b). Concentrations of ROS and RNS were measured either with colorimetric methods (as for nitrate, nitrite, and hydrogen peroxide) or by spin-trap-enhanced EPR spectroscopy (as for hydroxyl and superoxide anion radicals). Samples were taken of 5 mL of DPBS solution subjected to 180 s of plasma treatment as measured and summarized (a) (37,38). We identified an activation of several transcription factors, e.g., Nrf2 and p53 via MAPK signaling pathways, and a VEGF release in human HaCaT cells (12,33,40). Cold plasma plays a crucial role in the cellular response to oxidative stress via Nrf2 pathway as well as in the process of proliferation and migration ex vivo by upregulation of growth factor expression like VEGF, CSF2, etc. Furthermore, plasma-induced upregulation of CHAC1 may play a role in the unfolded protein response, and in regulation of glutathione levels and oxidative balance in the cell. Moreover, OSGIN1 is a key regulator of both inflammatory and antiinflammatory molecules (b). Biophysical Journal 2017 112, 2397-2407DOI: (10.1016/j.bpj.2017.04.030) Copyright © 2017 Biophysical Society Terms and Conditions