Fig 2: Activity-induced increase of pERK is abolished in Ptpn11D61Y neurons and can be restored upon MEK1 inhibition.Staining (A) and quantification (B) of pERK in nuclei (marked by DAPI) of excitatory neurons revealed elevated basal nuclear pERK levels in Ptpn11D61Y neurons. The basal levels of nuclear pERK significantly differ between the genotypes (unpaired t-test, ###p=0.001). The stimulation of neuronal activity induces a rapid increase in the nuclear pERK level (in relation to the basal levels) in control neurons but not in those from Ptpn11D61Y mice. The inhibition of MEK1 using SL327 for 24 h prior to the stimulation normalized the elevated basal pERK in nuclei of Ptpn11D61Y neurons and fully restored the activity-induced increase of nuclear pERK. The identical treatment affected neither the basal nuclear pERK levels nor their stimulation-induced increase in control neurons. Data are presented as mean ± SEM; numbers in columns indicate the numbers of analyzed cells. Significance is assessed using unpaired t-test and one-way ANOVA followed by Bonferroni´s multiple comparison test (**p=0.01, ***p=0.001).

Fig 3: The muscle of mice exposed to chemotherapy exhibits up-regulation of ERK1/2 and p38 MAPKs and down-regulation of mitochondrial proteinsRepresentative Western blotting (A) and quantification (B) for p-STAT3, STAT3, p-AKT, AKT, pMEK1/2, MEK1/2, p-ERK1/2, ERK1/2, p-p38, p38, PGC-1a, PGC-1ß, Cytochrome C (Cyt-C), LC3B, Beclin-1 and p62 in muscle protein extracts from mice exposed to chemotherapy. Levels of phosphorylated proteins were normalized to the respective total protein expression. Tubulin was used as loading control. Data are expressed as Fold change vs. Vehicle and reported as means ± SEM. Significance of the differences: *p < 0.05; **p < 0.01; ***p < 0.001 vs. Vehicle.

Fig 4: The influence of ERK1/2 and p38 inhibition on ozone-associated IL-8 induction. To determine whether the activation of the MAP kinases ERK1/2 and p38 were required for ozone-mediated IL-8 induction, inhibitors of these kinases (250 nM SCH772984 and LY2228820, respectively) were added to cell media two hours prior to ozone exposure. (A) The phosphorylation of downstream targets of ERK1/2 and p38, RSK and MK2, respectively, were examined to verify kinase inhibition. Extract from phBECs stimulated with 5 μg/mL LPS for four hours was used as a positive control. The LPS positive control for each mark was run on the same gel, immunoblotted, and imaged at the same time as the blots in the adjacent row. The images were cropped to allow the LPS treatments to be shown in the same lane. (B) Seven donors were used for drug treatments, four of which were high responders (shown above) and three low responders (Figure S1). IL-8 inductions were normalized to a 0.2% DMSO filtered air vehicle control. IL-8 expression was compared between the ozone-vehicle control (O3−V) and all other treatments via 2-way ANOVA. *p < 0.05.

Fig 5: Regulation of the MEK-ERK signaling pathway in 14.5 dpc hearts from two different aMEK1 transgenic models. Despite evidence of aMEK1 expression in the atria of both TAMEK/MH DTg and MEK1 single Tg mice, increased ERK1/2 phosphorylation was documented only in the DTg. *P < 0.05. P-ERK1/2 = Phospho-ERK1/2; A1/V1 = pooled atrial and ventricular tissues, respectively, from TAMEK/MH DTg; A2/V2 = pooled atrial and ventricular tissues, respectively, from TAMEK/MH WT sibling controls; A3/V3 = pooled atrial and ventricular tissues, respectively, from MEK1 single Tg; A4/V4 = pooled atrial and ventricular tissues, respectively, from MEK1 WT sibling controls. Original blots available in Additional file 7 S7