Demonstrated are results from one representative experiment (C). (B-C). Simultaneously to the animal experiment, cells were cultured in the absence of antibiotic selection to confirm stability of Pim-3 overexpression during the three-week test period (D).(TIF) pone.0130340.s001.tif (2.6M) GUID:?8529FE8D-CAD5-457B-AB58-A4C9FEB5A3B0 S2 Fig: Both DHPCC-9 and BA-1a Pim inhibitors decrease migration and viability of stable Pim-overexpressing PC-3 cells. Cell motility of stable control (C), Pim-1 (P1) or Pim-3 (P3) overexpressing Personal computer-3 prostate malignancy cells was analysed by wound healing assays. Cells were cultured on 24-well plates until confluency, after which wounds were scratched with 10 l pipette suggestions. Cells were treated with 0.1% DMSO or DMSO-dissolved Pim inhibitors and samples were imaged and analysed at 0 and 24 h time-points. Demonstrated are representative images along with average ideals from cells treated with either DHPCC-9 (A) or BA-1a (B). After 24 and 72 hours, viability of the cells was analysed by MTT assays. Demonstrated are average OD570 ideals from triplicate samples from one representative experiment (C). For each assay, at least three independent experiments were carried out with highly Aclacinomycin A related results.(TIF) pone.0130340.s002.tif (2.9M) GUID:?C3E11596-DDB8-4913-BBF2-2E486AB094B7 S3 Fig: DHPCC-9 tolerance in zebrafish embryos. Zebrafish embryos were treated at 6 h post-fertilization and analysed at 50 h post-fertilization. Demonstrated is average survival in two experiments (A), and body curvatures (B-C) as well as pericardial sac sizes (D) in one experiment with representative images to visualize the perspectives and the pericardiac Aclacinomycin A sac indicated by an arrow.(TIF) pone.0130340.s003.tif (662K) GUID:?441825EE-4C0A-4590-B0A3-641ED2F152D8 S4 Fig: Mouse weight gain during toxicity testing. Aclacinomycin A White colored male or female mice were treated with numerous concentrations of either DMSO (A) or DMA (B-C) diluted Pim inhibitors and adopted up for indicated time-periods to gain information about the possible cytotoxicity of the compounds.(TIF) pone.0130340.s004.tif (575K) GUID:?B3E1FCC5-4FA0-43A5-81CC-A5677BBE0043 S5 Fig: Mouse weight gain during the second orthotopic experiment. Stable control (C) or Pim-1 (P1) or Pim-3 (P3) overexpressing Personal computer-3 cells were orthotopically inoculated into nude mice. Mice were treated with DMSO or DMA like a control or with Pim inhibitors DHPCC-9 or BA-1a. Demonstrated is the average mouse weight gain in each group during the test period.(TIF) pone.0130340.s005.tif (344K) GUID:?85B6A6E5-D217-41E9-89B2-EE6E55E2941A S6 Fig: Fluorescent imaging of the second orthotopic set tissue samples. At the second orthotopic arranged, tumors and cells samples were fluorescently imaged to obtain information within the Tomato-derived transmission of stably transfected Personal computer-3 cells (Mock = C, Pim-1 = P1, Pim-3 = P3). Mice with control or Pim-3-overexpressing tumors were treated with 50 mg/kg of DHPCC9 in DMSO or 20 mg/kg of BA-1a Muc1 in DMA or vehicles only. After approximately three weeks, mice were sacrificed and cells were imaged. In each animal, transmission intensity was normalized relating to background transmission given by a kidney. Lymph nodes are pointed out by arrows. Demonstrated are images from tumors and collected tissue samples (A). After detection of metastases in the lymph node and lung sections, the typical areas of the metastases and the average necrotic areas in them were analysed. Demonstrated are areas as well as the number (n) of mice with metastases in control treated and DHPCC-9 treated animals (B).(TIF) pone.0130340.s006.tif (2.2M) GUID:?863E54B2-B83A-4CE4-987B-FA244A88C4BE S7 Fig: V5-immunostaining of xenografted cells within orthotopic tumors and their lymph node metastases. Paraffin-embedded cells Aclacinomycin A sections from the second orthotopic set of tumors (Mock = C, Pim-1 Aclacinomycin A = P1 and Pim-3 = P3), their surrounding mouse cells and one control tumor (Neg. Ctrl) were stained with anti-V5 antibody. Demonstrated are representative images from V5-positive ornegative samples.(TIF) pone.0130340.s007.tif (6.2M) GUID:?780E5757-760A-4632-9C2D-C8447C2783D2 S8 Fig: Pim-1 and Pim-3 increase and DHPCC-9 decreases CXCR4 phosphorylation in PC-3 cells. Personal computer-3 cells transiently overexpressing an empty vector (C), Pim-1 (P1), Pim-2 (P2) or Pim-3 (P3) were treated with DMSO or 10 M DHPCC-9 for 24 hours. CXCR4 phosphorylation was recognized by phospho(Ser339)-CXCR4 antibody, after which the transmission intensity was compared to the intensity of the CXCR4 transmission. Pim overexpression.