e harvested and lysed in RIPA buffer supplemented with protease inhibitors. For each experiment, samples were normalized for protein concentration and equal amounts of material in Laemmli sample buffer were subjected to SDS-PAGE and transferred to nitrocellulose filters. For immunoblotting the HPA anti-FHOD1 antibody was incubated overnight at 1:2000 dilution in BSA/ TBS/Tween 0.1%, followed by secondary antibody. Bound proteins were detected by enhanced 18998663 chemiluminescence. In the peptide competition experiment the antibody was first incubated with a 100-fold molar excess of GST-FHOD1 fusion protein containing the antigenic epitopes for 1hour, or with GST as a control. Protein loading was checked by immunoblotting with an a-tubulin antibody. Epithelial/ mesenchymal transdifferentation was checked by immunoblotting with anti-E-cadherin and anti N-cadherin antibodies. Northern blot analysis was performed as described. The sequence of the probe included most of the antibody epitope. Transcriptomic microarray data and quantitative real-time-PCR Gene expression was analysed using the Illumina HumanHT-12 v4 Expression BeadChip at the Finnish Microarray and Sequencing Centre, Turku Center for Biotechnology. Total RNA was extracted from cultured cells using RNeasy Mini kit according to the 606143-89-9 manufacturer’s protocol and processed to cDNA with cDNA synthesis kit. The array-based data on cell lines has been loaded to ArrayExpress. TaqMan qRT-PCR was performed with an Applied Biosystems 7900HT instrument. Probes and primers were from Oligomer, Helsinki, Finland. Quantitation was carried out with RQ manager 1.2 software using the DDCT method. Three replicate samples were studied for detection of target mRNA expression and b-actin used as an endogenous control. The quantities were expressed as an n-fold difference relative to the UT-SCC-43A cell line. The results are presented as means 6 SD. Statistical analyses were performed using Student’s t-test and Pearson’s correlation coefficient, unless otherwise indicated. Gene specific primers were: DIAPH1, FHOD1, FHOD3, FMNL3. Inhibition of MAPK/ERK and PI3K signalling pathways UT-SCC-43B cells were plated in complete medium and cultured for 24 hours before medium was replaced by serum free medium and incubated overnight. Next, the cells were incubated for 4 days in 10mM MEK 1/2 inhibitor U0126 or PI3 kinase inhibitor LY294002 in 1% serum medium for U0126 and 10% serum medium for LY 294002. Control cells were treated with the same volume of the vehicle. After incubation, the efficacy of MAPK pathway inhibition was checked by immunoblotting as described above with 1:1000 dilutions of 
polyclonal anti-Phospho-p44/42 MAPK and polyclonal anti-ERK 2 antibody. PI3K pathway inhibition was checked by immunoblotting with monoclonal anti-Phospho-Akt , monoclonal anti-Akt . Gene silencing FHOD1 expression was transiently knocked 2181489 down in MDAMB-231, TIME and UT-SCC-43B cells using SMARTpool siRNA. Non-targeting Pool siRNA was used as a control. Cells were transfected with Dharmafect 1 according to manufacturer’s instructions. FHOD1 levels were examined in cell lysates 72 hours after transfection by immunoblotting. FHOD1 antibodies A FHOD1 rabbit anti-human polyclonal monospecific antibody was produced by the Human Protein Atlas program , and is currently available to the public. Further antibody characterisation is described in the Results section. In some experiments, In silico transcriptomics analysis The GeneSapiens dat
