*, p < 0,05). AKT3 knockdown increases the expression of the promigratory protein S100A4 Next, the phosphorylation of AKT at serine residue 473 (S473) and threonine residue 308 (T308), reflecting the activation status of AKT, were analyzed. by lentiviral transduction using AKT isoform specific shRNAs. Knockdown efficacy was confirmed by Western blot analysis. (B)-(C) Analysis of cell migration using scratch assay technique. A confluent monolayer was scratched using a 200l pipette tip and cell migration was analyzed using time lapse video microscopy, as described in section 3.6. Mean single cell velocity of Hep3B control and AKT isoform knockdown cells is usually given in (B). One representative experiment out of three is usually shown (Bars: SD. **, p < 0,01. ***, Pimobendan (Vetmedin) p < 0,001). (C) Representative images of the scratch assay after 0, 12, 24 and 36 hours are shown. (D) Proliferation was analyzed by manual cell counting over four consecutive days, performed in triplicates. Proliferation is usually shown Pimobendan (Vetmedin) as relative cell count normalized to day 0 (Bars: SD, n.s., p>0,05).(TIF) pone.0146370.s002.tif (2.4M) GUID:?62962B7E-51E7-4047-B553-18854BCCA3BB S1 Video: Time lapse video of migrating SCR controls vs. AKT2,3 double knockdown MDA-MB-231 cells. Representative live cell imaging video of migrating MDA-MB-231 double knockdown cells. A confluent monolayer was scratched using a 200l pipette tip and cell migration was analyzed using time lapse video microscopy. Pictures were taken every five minutes to generate videos.(MOV) pone.0146370.s003.mov (8.4M) GUID:?E4816B69-ECA4-41B6-BE36-42CA617D275B S2 Video: Time lapse video of migrating SCR controls vs. AKT1,3 double knockdown MDA-MB-231 cells. Representative live cell imaging video of migrating MDA-MB-231 double knockdown cells. A confluent monolayer was scratched using a 200l pipette tip and cell migration was analyzed using time lapse video microscopy. Pictures were taken every five minutes to generate videos.(MOV) pone.0146370.s004.mov (9.1M) GUID:?8D6F3A71-F912-43D6-9E12-BA005AD57598 S3 Video: Time lapse video of migrating SCR controls vs. AKT1,2 double knockdown MDA-MB-231 cells. Representative live cell imaging video of migrating MDA-MB-231 double knockdown cells. A confluent monolayer was scratched using a 200l pipette tip and cell migration was analyzed using time lapse video microscopy. Pictures were taken every five minutes to generate videos.(MOV) pone.0146370.s005.mov (7.7M) GUID:?4D7D58BF-47EB-4B21-B7B1-C711278CFE92 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Background Treatment of breast cancer patients with distant metastases represents one of the biggest challenges in todays gynecological oncology. Therefore, a better understanding of mechanisms promoting the development of metastases is usually of paramount importance. The serine/threonine kinase AKT was shown to drive cancer progression and metastasis. However, there is emerging data that single AKT isoforms (i.e. AKT1, AKT2 and AKT3) have different or even opposing functions in the regulation of cancer cell migration chemotaxis measurement [28]. FCS with a final concentration of 10% (v/v) was used as a chemoattractant. Cells were seeded at high Pimobendan (Vetmedin) density (3×10^6/ml) into the observation area of the 3D chemotaxis slides, and reservoirs were filled according to the instructions provided by the manufacturer. Time lapse images were recorded as described above. Velocity and euclidean distance were analyzed using ImageJ Rabbit Polyclonal to GCNT7 software. Subcutaneous tumor xenograft model and analysis This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All experimental protocols were approved by local authorities (Ministry of Health and Consumer Protection, Hamburg, Germany, Permit Number G11/12)..