. Comparisons between two groups were analyzed via t test, and values of P,0.05 were considered to be significant.For P-SRSF2, tumors were sub-divided in three classes. For SRPK1 and SRPK2, distinct cut-off were chosen for adenocarcinoma and squamous cell carcinoma based on the different intensity of staining observed in alveolar type II pneumocytes or bronchial cells. In this study, we investigated the status of SRSF1, SRSF2 and its phosphorylated form P-SRSF2, as well as PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19828691 of SRPK1 and SRPK2 in a series of 107 NSCLC, including 54 adenocarcinoma and 53 squamous cell carcinoma. Our results reveal a global overexpression of these splicing regulators in NSCLC compared to normal lung tissues, that correlate with more aggressive clinico-pathological features in ADC. In agreement with these data, we provide evidence that overexpression of SRSF1 in cellular models derived from human lung adenocarcinoma leads to a more aggressive phenotype with activation of p42/44MAPK and AKT signaling 487-52-5 biological activity pathways, increased colony formation in soft agar, epithelial to mesenchymal transition and resistance to carboplatin and paclitaxel.They were transfected either with a pcDNA3 empty vector or with a pcDNA3-SRSF1-Myc tagged vector using Fugene 6 reagent. H358 transfected cells were grown in RPMI-1640 medium containing 800 mg/ml geneticin for at least 4 weeks in order to select stable transfectants. Single colonies were isolated and expanded for further analyses. H1299 and H2170 transfected cells were selected during 6 days 
using 800 mg/ml G418 before analysis of EMT markers. U0126, wortmaninn, etoposide, paclitaxel and carboplatin were all purchased from Sigma. Cytotoxic experiments were performed using the methylene blue colorimetric assay as previously described. Anchorageindependent growth was determined by assaying colony formation in soft agar. Briefly, H358-Ctl and H358-SRSF1 cells were resuspended in RPMI containing 10% FCS and 0.3% agar noble and plated in quadriplicate on a firm 0.6% agar noble base in 12-well plates. Colonies of cells were allowed to grow 21 days in a 37uC and 5% CO2 incubator. Colonies were then observed and counted under an inverted microscope. The colony formation assay was performed in triplicate. performed using the LightCyclerH 480 Real-Time PCR system. One microgram of total RNA was subjected to cDNA synthesis with Superscript III First-Strand Synthesis SuperMix for qPCR and subsequently amplified during 45 PCR cycles using GoTaqH qPCR Master Mix. Primers used for the detection of GAPDH and SRSF2 mRNAs were detailed previously. Primers for amplification of SRSF1, SRPK1 and SRPK2 mRNAs were purchased from SA Biosciences. Relative gene expression was calculated for each sample, as the ratio of specific target gene to GAPDH gene, thus normalizing the expression of target gene for sample to sample differences in RNA input. Statistical Analysis The staining scores were compared in different categories using Fisher’s exact test and Mann-Whitney test. All tests were twotailed and p values,0.05 were considered significant. The statistical analyses were done using Statview software. Results Overexpression of SRSF1 Protein is Associated with Extensive Stage Tumors We first analyzed the status of SRSF1 protein in our series of lung tumors. Of note, analysis of phospho-SRSF1 protein was not possible due to the lack of a specific anti-phospho-SRSF1 antibody. SRSF1 was slightly expressed in normal lung epithelium adjacent to tumor cell