The percentage of cells containing invadopodia was counted from 10 different fields in each group. Differences between groups were tested for significance by ANOVA in both experiments. Immunofluorescence microscopy and quantification of F-actin 16260133 UT-SCC-43A and UT-SCC-43B cells were cultured on gelatine-coated coverslips, and fixed with 4% paraformaldehyde for 15 min. The cells were permeabilised with cold acetone for 4 min. After 30 min in 1% bovine serum albumin in PBS, the cells were incubated for 1 h with the FHOD1 antibody, followed by a secondary antibody, Alexa 568-conjugated goat antirabbit. F-actin was visualized with Alexa 488-conjugated phalloidin. The mounting media contained DAPI for staining nuclei. Invadopodia were visualised by immunofluorescence staining with anti-cortactin 1:200. Plasma cells were identified with antiCD138 mAb. Alexa Fluor 568-conjugated goat anti-mouse was used as secondary antibody. The cells were imaged with immunofluorescence microscopy or a Zeiss LSM 510 Meta confocal microscope. Image J 1.42q software was used in picture analyses. Quantification of F-actin in UT-SCC43B cells was done by analysing Alexa 488-conjugated phalloidin staining from confocal images of 20 cells from each experiment. Mean fluorescent intensities were measured from the cell cytoplasm. The significance of the experiment was calculated using Students independent samples t-test. Results Transcriptional regulation of formins during cancer-associated EMT We used three model cell lines to evaluate alterations in formin expression during cancer-associated EMT. UT-SCC-43A is an oral SCC cell line from primary tumour and UT-SCC-43B is a line from the same tumour recurring after surgery and radiotherapy. The recurring tumour has undergone a spontaneous EMT, as demonstrated by several markers. The third cell line, 43ASNA, was established by transfecting UT-SCC-43A cell with Snail to induce EMT. Transcriptomic analysis revealed that the mesenchymal cell lines UT-SCC-43B and 43A-SNA have 35 up-regulated genes and 153 down-regulated genes in common. In UT-SCC43B and 43A-SNA, established EMTmarkers such as N-cadherin, vimentin and collagens were upregulated, whereas epithelial markers such as Wound healing and invasion assays Migration of UT-SCC-43B cells treated with FHOD1 or nontargeting siRNAs was studied on gelatine-coated 24-well-plates. A wound was created by manually scraping the monolayer 
of cells with a 10 ml GW 5074 pipette tip. The cells were washed with PBS, and filtered medium was added. An image of cells migrating into the wound was taken at 10 min intervals for 24 h. ImageJ software was used for measuring the wound area 9521749 at 1 h intervals. The wounds were analysed using repeated measurements analysis of variance. Time was held as repeated effect and Role of FHOD1 in EMT E-cadherin, keratins, integrins and laminins were simultaneously down-regulated indicating that the transcriptomic alterations are consistent with EMT. Of the 13 formin family members included in the array, two were consistently up-regulated and four were down-regulated during EMT. The most significant up-regulation was seen with FHOD1, which was increased 2.3-fold in UT-SCC-43B and 3.2fold in 43A-SNA as compared with UT-SCC-43A. The significant transcriptional differences were further verified by qRT-PCR, which demonstrated upregulation of formins FHOD1 and FMNL3 and downregulation of FHOD3 and DIAPH1 in both UT-SCC-43B and 43A-SNA cells. Characterization of human FHO The percentage of cells containing invadopodia was counted from 10 different fields in each group. Differences between groups were tested for significance by ANOVA in both experiments. Immunofluorescence microscopy and quantification of F-actin UT-SCC-43A and UT-SCC-43B cells were cultured on gelatine-coated coverslips, and fixed with 4% paraformaldehyde for 15 min. The cells were permeabilised with cold acetone for 4 min. After 30 min in 1% bovine serum albumin in PBS, the cells were incubated for 1 h with the FHOD1 antibody, followed by a secondary antibody, Alexa 568-conjugated goat antirabbit. F-actin 23570531 was visualized with Alexa 488-conjugated phalloidin. The mounting media contained DAPI for staining nuclei. Invadopodia were visualised by immunofluorescence staining with anti-cortactin 1:200. Plasma cells were identified with antiCD138 mAb. Alexa Fluor 568-conjugated goat anti-mouse was used as secondary antibody. The cells were imaged with immunofluorescence microscopy or a Zeiss LSM 510 Meta confocal microscope. Image J 1.42q software was used in picture analyses. Quantification of F-actin in UT-SCC43B cells was done by analysing Alexa 488-conjugated phalloidin staining from confocal images of 20 cells from each experiment. Mean fluorescent intensities were measured from the cell cytoplasm. The significance of the experiment was calculated using Students independent samples t-test. Results Transcriptional regulation of formins during cancer-associated EMT We used three model cell lines to evaluate alterations in formin expression during cancer-associated EMT. UT-SCC-43A is an oral SCC cell line from primary tumour and UT-SCC-43B is a line from the same tumour recurring after surgery and radiotherapy. The recurring tumour has undergone a spontaneous EMT, as demonstrated by several markers. The third cell line, 43ASNA, was established by transfecting UT-SCC-43A cell with Snail to induce EMT. Transcriptomic analysis revealed that the mesenchymal cell lines UT-SCC-43B and 43A-SNA have 35 up-regulated genes and 153 down-regulated genes in common. In UT-SCC43B and 43A-SNA, established EMTmarkers such as N-cadherin, vimentin and collagens were upregulated, whereas epithelial markers such as Wound healing and invasion assays Migration of UT-SCC-43B cells treated with FHOD1 or nontargeting siRNAs was studied on gelatine-coated 24-well-plates. A wound was created by manually scraping the monolayer of cells with a 10 ml pipette tip. The cells were washed with PBS, and filtered medium was added. An image of cells migrating into the wound was taken at 10 min intervals for 24 h. ImageJ software was used for measuring the wound area at 1 h intervals. The wounds were analysed using repeated measurements analysis of variance. Time was held as repeated effect and Role of FHOD1 in EMT E-cadherin, keratins, integrins and laminins were simultaneously down-regulated indicating that the transcriptomic alterations are consistent with EMT. Of the 13 formin family members included in the array, two were consistently up-regulated and four were down-regulated during EMT. The most significant up-regulation was seen with FHOD1, which was increased 2.3-fold in 18753409 UT-SCC-43B and 3.2fold in 43A-SNA as compared with UT-SCC-43A. The significant transcriptional differences were further verified by qRT-PCR, which demonstrated upregulation of formins FHOD1 and FMNL3 and downregulation of FHOD3 and DIAPH1 in both UT-SCC-43B and 43A-SNA cells. Characterization of human FHO