Validation of the MAPK role in DFNA5-related cell death in HEK293T cells
The data generated by the transcriptomic analysis in HEK293T cells were validated by real-time rtPCR of newly collected RNA samples. EGR1 and FOSB gene expression was investigated on different time-points ranging from 3 to 72 h post-transfection. As shown in Figure 2, significantly up-regulated EGR1 and FOSB gene expression was observed in cells transfected with mutDFNA5 from 12 to 18 h post transfection (p < 0.05) (Figure 2). Hence the data generated by the transcriptomic analyses were indeed confirmed by real time rtPCR as demonstrated by up-regulation of genes related to the MAPK pathway.
Figure 2  Increased EGR1 and FOSB gene expression in mutDFNA5-transfected HEK293T cells. RNA samples were collected from HEK293T cells transfected with either wtDFNA5 or mutDFNA5 and gene expression was measured by real-time rtPCR. Significantly increased expression was seen in mutDFNA5 at 12 h [p(egr1) = 0.000; p(fosB) = 0.006], 15 h [p(egr1) = 0.017; p(fosB) = 0.000] and 18 h [p(egr1) = 0.004; p(fosB) = 0.026] post-transfection. *p < 0.05; **p < 0.01; ***p < 0.001. CNRQ, calibrated normalized relative quantities.   After confirmation by real time rtPCR, the significance of the activated MAPK pathway was further validated by two independent experiments. To investigate the significance of the MAPK pathway, we wondered whether inhibition of the MAPK pathway would attenuate this mutDFNA5-induced growth defect.
Therefore, a specific JNK inhibitor, namely SP600125, was added, followed by a viability assay to determine the effect on cell survival. Different concentrations of the JNK inhibitor SP600125 were used to measure viability by flow cytometry (CyFlow ML, Partec, Germany) and these results were compared to untreated mutDFNA5-transfected HEK293T cells. Overnight treatment of the cells with different concentrations of SP600125 did not have any major effect on transfection efficiency, but significantly increased the viability of mutDFNA5-transfected cells. Although, addition of 12.5 and 25 μM SP600125 both significantly increased the viability with a p-value of 0.020 and 0.004 respectively SP600125 had the greatest effect with a concentration of 25 μM SP600125 since the viability was raised from 31.93 to 51.00% (Figure 3).
Figure 3  MAPK inhibitor effect on mutDFNA5 transfected HEK293T cells. MutDFNA5-transfected HEK293T cells were pretreated with different amounts of SP600125 (JNK inhibitor). Cell viability was measured and compared to untreated mutDFNA5-transfected HEK293T cells. *p < 0.05; **p < 0.01.   Next, to evaluate the effect of MAPK up-regulation on protein level, different MAPK proteins were studied by western blotting. There are three main MAPK pathways in human cell lines represented by the ERK, JNK, and p38 MAPK branch. Consistent with the results obtained by real time rtPCR and the viability assay, activation of the MAPK pathway proteins was also demonstrated by western blotting. Total protein lysates were collected from HEK293T cells 12 h post-transfection. Three phosphorylated (activated) and non-phosphorylated (not activated) proteins of the MAPK pathway were studied using six different antibodies. No differences were seen in the expression level of non-phosphorylated ERK and JNK (Figure 4A). Activation of JNK and to a minor extent of ERK (p42/p44) was seen upon mutDFNA5 transfection compared to control and wtDFNA5 (Figure 4B). The expression of p38 was also evaluated but no difference in protein expression was observed between mutDFNA5 compared to wtDFNA5 and control (data not shown). β-Actin was used as a loading control.
Figure 4  Activation of the MAPK pathway by mutDFNA5. (A) Western blot analysis of non- phosphorylated ERK (p42/p42) and JNK. No differences were seen in protein expression level of ERK and JNK between control, wtDFNA5 and mutDFNA5 transfected HEK293T cells. (B) Western blot analysis of phosphorylated, activated ERK (p42/p42) and JNK. Increased expression of JNK and to a lesser extent of ERK was seen in mutDFNA5 transfected HEK293T cells as compared to wtDFNA5 and control.   These results suggest that DFNA5 induces PCD mediated through activation of the MAPK pathways. Addition of a MAPK inhibitor partially attenuated the mutDFNA5-induced growth defect identifying the MAPK pathway as an early event in mutDFNA5-associated cell death.