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ation coming through the artificial membrane being less than that of the non-transfected Fig 1. Overexpression of SRPK1 and SRSF1 in glioma cells. Observed HA, U251 and A549 under fluorescence microscope by Hoechst staining of nuclei, immunostaining for SRPK1 and SRSF1, scale bars: 100m. Western blot results showed the levels of SRPK1 and SRSF1 in U251, U87 and HA cells, -Actin was used as an internal loading control. Bar graph showing summary analyses of SRPK1 and SRSF1 with U251 cells by qRT-PCR in normoxic and hypoxic condition, compared with HA cells respectively. These data indicate that SRPK1 is a cisplatin-sensitive gene, which might potentially play a role in glioma cells. To further evaluate temozolomide effected on the scramble or siSRPK1 U251 cells and investigated cisplatin in comparison with TMZ through flow cytometry. In order to quantify the magnitude of SRPK1’s ability to induce chemosensitivity, median lethal dose of DDP and TMZ was firstly identified. A range of 080 ug/ml of DDP was determined by MTT and half of cell viability was measured at a concentration of 20 ug/ml. Similarly, 0500 uM of TMZ was tested for its effects on half of U251 cell viability and the half lethal concentration was 125 uM of TMZ. Furthermore, siSRPK1 was added 24h prior to addition of the chemotherapeutics, and the results showed that the inhibited SRPK1 expression could decrease the cells apoposis caused by DDP and TMZ in normoxic condition. Interestingly, this role was reversed in hypoxic condition, especially for cells treated by DDP. To further validate SRPK1 impacts on the sensitivity of drugs in glioma cells, we studied the changes of SRPK1 and SRSF1 after cells treated by DDP and TMZ PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19834545 in normoxic and hypoxic condition at mRNA and protein levels. RT-PCR analysis was performed to detect the up-regulated SRPK1 mRNA levels that are presented in normoxia after DDP and TMZ treatment. SRSF1 mRNA was increased by TMZ MG516 cost treatment more than DDP treatment in normoxic condition. However SRPK1 mRNA levels were mildly elevated, but SRSF1 gene transcription was still inhibited by TMZ treatment in hypoxia. Western blot results showed that DDP treated cells could promote the release of SRPK1 and SRSF1 protein in nomoxia, while SRPK1 expression was inhibited and the expression of SRSF1 was increased in hypoxia. SRPK1 was obvious inhibited by TMZ treatment in nomoxia or hypoxia and had little impacts on the whole expression of SRSF1. Therefore, SRPK1 can be regarded as a cisplatin-sensitive gene but not as a temozolomide -sensitive gene. Fig 4. Downregulated SRPK1 increases chemotherapeutic drug resistance of glioma cells in normoxic and hypoxic condition. Viability of U251 cells exposed to different concentrations of DDP for 24,48,72 h. The results are presented as percentage. Cell viability was evaluated by MTT assay. Viability of U251 cells exposed to different concentrations of TMZ for 24,48,72 h. The results are presented as percentage. Cell viability was evaluated by MTT assay. Bar graph showing the different of cell apoptotic rates between the control and siSRPK1 U251 cells treated by DDP or TMZ in normoxic or hypoxic condition. U251 cells were transfected for 24h with scramble SRPK1 siRNAs and treated or not for an additional 48 h with 200 uM TMZ or 20ug/ml DDP. Cell apoptotic rates were analyzed by flow cytometry. http://www.jcancer.org Journal of Cancer 2013, Vol. 4 733 Fig 5. To explicit SRPK1 can increase DDP resistance of glioma cells in molecul