Otting. The pattern observed on the transcriptional level was confirmed by an enhanced expression of BBC3/Puma (Figure 7(c)), Gadd45 (Figure 7(d)), and CDKNA1/p21 (Figure 7(e)) when Bax remained unchanged (data notRelative phosphorylation level eight 20 six 4 two 15 10Oxidative Medicine and Cellular LongevityAcetylcholine estereas Inhibitors Reagents p-Erk2 p-Erk1 Erk2 Erk1 -Actin ctrl 20 60 180 ctrl 20 60 180 CAV2 Inhibitors Reagents Plasma treatment time (s) p-Erk2 p-Erkp-Jnk2 p-Jnk1 Jnk2 Jnk1 -Actin Plasma remedy time (s) p-Jnk2 p-Jnk(a)four 15 Relative phosphorylation level(b)p-p38 p38 -Actin ctrl 20 60 180 Plasma therapy time (s) ctrl 0.25 0.five 0.75 1h 3h 6h 24hp-Erk2 p-Erk1 Erk1/2 -Actin Incubation time following plasma treatment (h) p-Erk2 p-Erk(c)8 Relative phosphylation level 15 Relative phosphylation level(d)6p-Jnk2 p-Jnk1 Jnk2 Jnk1 -Actin ctrl 0.25 0.5 0.75 1 h three h 6 h 24 h Incubation time immediately after plasma therapy (h) p-Jnk2 p-Jnk1 ctrl 0.25 0.5 0.75 1h 3h 6h 24 h Incubation time immediately after plasma treatment (h)p-p38 p38 -Actin(e)(f)Figure 6: Plasma-induced activation of MAP kinase signaling in HaCaT keratinocytes. Displayed are relative phosphorylation levels of Erk (p-T202/Y204, a), Jnk (p-T183/Y185 (b)), and p38 (p-T180/Y182, (c) kinases immediately after distinct therapy times. Lower panels showed the outcomes for time course of relative phosphorylation levels soon after 180 s of plasma treatment for Erk (d), Jnk (e), and p38 (f). Each and every expression was normalized to total protein expression. Representative blots are shown. Information are presented as imply + S.D. of two analyses. The x-axis represents remedy time (a ) or incubation following plasma therapy (d ). Statistical analysis was completed employing one-way ANOVA with Dunnett corrections for various comparisons to untreated, normalized control ( p 0 05, p 0 01, p 0 001).Oxidative Medicine and Cellular LongevityRelative phosphorylation level 20 15 10 GADD45 five CDKN1A p-Hsp27 Hsp27 -Actin ctrl 20 60 180 Plasma treatment time (s) 1 3 6 12 24 Incubation time right after plasma therapy (h) GAPDH BAX(a)8 Relative gene expression six 4 two BAX BBC3 GADD45 CDKN1A Normalized expression eight 6 4 two PUMA(b)GADDctrl 20 60 180 20 60 180 20 60 180 20 60 180 Plasma treatment time (s) ctrl 20 60 180 ctrl 20 60 180 Plasma therapy time (s)Puma/GADD45 -Actin(c)p21 expression (normalized) p21 expression (normalized) 25 20 15 ten five 25 20 15 10(d)p2 -Actin ctrl 20 60 180 Plasma therapy time (s) ctrl 0.25 0.5 0.75 1 3 six 24 Incubation time immediately after plasma remedy (h)p21 -Actin(e)(f)Figure 7: Cold plasma-induced influence on important p53 downstream targets. The total amount plus the phosphorylated form of the stressrelated protein HSP27 was drastically enhanced following plasma therapy (a). Agarose gel showing semiquantitative PCR of BAX, BBC3, GADD45, and CDKN1A immediately after 1 to 24 h posttreatment of HaCaT cells with 60 s plasma (b). mRNA copy numbers of all four targets have been measured six h immediately after plasma remedy by qPCR and normalized for the relative gene expression (CT values on a log2 scale) (c). Puma (protein of BBC3), Gadd45 (d), and p21 (e) expression was significantly enhanced following plasma exposure (180 s). p21 (protein of CDKN1A) improved till six hours just after plasma therapy, declining afterwards (f). Representative blots are shown. The x-axis represents treatment time (a, c, d, and e) or incubation just after plasma remedy (b, f). Information are presented as imply + S.D. of two (c, d) or three (b) analyses. Statistical analysis was performed employing one-way ANOVA with Dunnett corrections for a number of comparisons to untrea.