HEK293T cells

Cell culture and growth conditions
The microarray experiments in human cell lines were performed using Human Embryonic Kidney 293T cells (HEK293T). HEK293T cells were subcultured in 60 mm dishes at a density of 2 × 106 cells in Dulbecco's modified Eagle's medium containing 4500 mg/L glucose supplemented with 10% (v/v) fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine. Overnight cultures were transfected with either control (empty EGFP vector), wtDFNA5 or mutDFNA5 using lipofectamine. Six hours post transfection, cells were harvested using Triple-x reagent for RNA extraction. All products for human cell line cultures were obtained from Invitrogen (San Diego, CA, USA). Full-length cDNA of either wtDFNA5 or mutDFNA5 was isolated and amplified as previously described (Gregan et al., 2003).

RNA extraction
An RNeasy mini kit was used for RNA extraction from transfected HEK293T cells (Qiagen, Hilden, Germany) at different time-points. For the microarray experiment, RNA was extracted 12 h post-transfection. For the gene analysis by real time rtPCR, RNA was extracted at either 3, 6, 12, 15, 18, 20, or 24 h post-transfection. For the microarray experiment, the integrity of the resulting RNA was checked on an automated Experion electrophoresis system (Biorad, Hercules, CA, USA).

Microarray design and analysis
The “Totalprep RNA Amplification” kit was used to amplify the RNA samples (Illumina, Ambion, Austin, TX, USA). Doublestranded-cDNA was generated from the mRNA fractions followed by an in vitro transcription reaction which produced cRNA strands containing biotin-UTP nucleotides. Seven Hundred Fifty nano gram of the resulting cRNA samples were hybridized to an Illumina human HT12v3 beadchip (Illumina, San Diego, CA, USA).
Six independent biological replicates were used for either wtDFNA5- or mutDFNA5-transfected HEK293T cells and loaded on the chip. Overnight hybridization at 58°C was followed by washing and streptavidin-Cy3 dye labeling (Amersham, Buckinghamshire, England). An Illumina Iscan equipped with Iscan control software was used to measure the intensity values and XY coordinates for every probe on the array. The resulting data files were then analyzed using the R package “Beadarray v1.14.0” (Dunning et al., 2007) followed by a quality control and quantile normalization. LIMMA v3.2.1 was used for the further analysis of the normalized intensity values to determine the differentially expressed genes (Smyth, 2004).
All the (raw) data files have been completely uploaded to the gene expression omnibus and have been stored under accession number GSE70169.

Real time rtPCR
To confirm the results obtained from the microarray data in HEK293T cells, gene expression was studied in human HEK293T cell lines using Power SYBR Green RNA-to CT 1 Step Kit (Invitrogen, San Diego, CA, USA). Each reaction mixture contained 200 nM final primer concentration (primer pairs are shown in Supplemental Data Table 1) and 30 ng RNA template. All reactions were performed in triplicate on a LightCycler 480 system (Roche, Basel, Switzerland) and resulting data were analyzed by Qbase plus (Biogazelle, Ghent, Belgium). Three housekeeping genes were used each time as a reference, namely GAPDH, RPL13A, and YWHAZ.

Western blot analysis
For western blotting, cells were lysed using RIPA buffer (25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (Pierce, Rockford, IL, USA) containing a PhosSTOP Phosphatase Inhibitor Cocktail Tablet (Roche), an EDTA-free protease tablet and 10 μl (25 units/μl) benzonase (70746-4 Novagen®, Merck Millipore, Darmstadt, Germany). Transfected HEK293T cells were lysed for 20′ at 4°C and centrifugated at 2000 g at 4°C to obtain protein lysates.
Proteins were electrophoretically separated and blotted onto a nitrocellulose membrane (Whatman, Kent, UK). This membrane was blocked for 1 h in 5% non-fat dry milk and afterwards incubated overnight (4°C) in one of the following primary antibodies: anti-phospho-SAPK/JNK (Thr183/Tyr185, #4668), anti-phospho-p44/42 (ERK1/2, #4370), anti-SAPK/JNK (#9252), anti-p44/42 (ERK1/2, #9102) (Cell Signaling Technologies, MA, USA), or anti-β-Actin (A5316, Sigma Aldrich, MO, USA). After washing, the membranes were incubated with either a secondary goat anti-rabbit (ab6721, Abcam, Cambridge, UK) or sheep anti-mouse (NA931, GE Healthcare, Buckingmersham, UK) antibody. Finally, the corresponding proteins were visualized using Enhanced ChemiLuminescence Western Blotting Substrate (Thermo Scientific, IL, USA).

MAPK inhibition
The SP600125 JNK inhibitor was used to inhibit the MAPK pathway. Twelve hours post-transfection, HEK293T cells were incubated with 25 μM for 12 h. Next, cells were collected and viability was measured by flow cytometry (CyflowML, Partec, Germany) using propidium iodide as a cell death marker.