Supplementary MaterialsS1 Fig: Supporting information for Fig 3. (E) or CPI-613 and chloroquine (F) was analyzed with a Guava EasyCyte cell analyzer.(TIF) pone.0198940.s002.tif (218K) GUID:?95579659-9A97-4E2B-B904-14939CA0DEF3 S3 Fig: Supporting information for Fig 3. A combination of CPI-613 and chloroquine significantly suppressed tumor growth in an orthotropic metastatic tumor model of CCS. A: Intraperitoneal administration of CPI-613 (25 mg/kg) and chloroquine (50 mg/kg) significantly decreased tumor growth at the injection site and reduced the Staurosporine biological activity metastasis of HS-MM cells in SCID-beige male mice. Arrow indicates a day of injection of CPI-613 and chloroquine (two times weekly). B: Total weights of collected, disseminated mesenteric tumors after seven days from your last CPI-613 and chloroquine injections. Data are expressed as means SD (n = 5). Students 0.01). C: Representative mice are shown. Note the reduction in metastasis of CPI-613 and chloroquine treated HS-MM cells (indicated as CPI-613) compared to those of control mouse. White arrow indicates the distant metastasis.(TIF) pone.0198940.s003.tif (226K) GUID:?C5E4137A-AF54-42FA-A78E-93D602F5A783 S4 Fig: Supporting information for Fig 3. Expression of the EWSR1-ATF1 fusion transcript. Expression of the fusion transcript was observed in distant metastasis to the lung (indicated as lung), ascites, and the primary injected site tumor of all five HS-MM transplanted SCID-beige mice (numbered as 1, 2, 3, 4, and 5). Image of agarose gel following electrophoresis with HINDIII DNA size marker.(TIF) pone.0198940.s004.tif (105K) GUID:?0C6466EE-D70B-459D-927A-CEC2172DDDCF S1 Table: Supporting information for Fig 4A. Longest diameter, shortest diameter, and calculated tumor volumes.(XLSX) pone.0198940.s005.xlsx (13K) GUID:?E0E36A7D-803F-4210-B153-F4C048AC3170 S2 Table: Supporting information for Fig 4B. Total weights of collected, disseminated mesenteric tumor.(XLSX) pone.0198940.s006.xlsx (8.7K) GUID:?5E4AEB1C-0CBC-4F7B-9631-FD43866F6B45 S3 Table: Supporting information for S3A Rabbit polyclonal to ACTG Fig. Tumor volumes of control and CPI613-Chloroquine treated mice.(XLSX) pone.0198940.s007.xlsx (9.3K) GUID:?A1DA52AA-87F1-45E2-9AF1-9CF8FB59DBF4 S4 Table: Supporting information for S3B Fig. Total weights of collected, disseminated mesenteric tumor.(XLSX) pone.0198940.s008.xlsx (9.9K) GUID:?C96F722D-E69A-437F-88CA-911898693083 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Clear cell sarcoma (CCS) is an aggressive type of soft tissue tumor that is associated with high rates of metastasis. In the present study, we found that CPI-613, which targets tumorous mitochondrial energy metabolism, induced autophagosome formation followed by lysosome fusion in HS-MM CCS cells fusion gene [12]. Cells were cultured with Dulbeccos altered Eagles medium (DMEM; Gibco Life Technologies, Grand Island, NY, USA) made up of Staurosporine biological activity 10% heat-inactivated fetal bovine serum. CPI-613 and chloroquine, autolysosome detection, and double staining with annexin V and propidium iodide CPI-613 and chloroquine were purchased from AdooQ BioScience (Irvine, CA, USA) and Nacalai Tesque (Tokyo, Japan), respectively. Necrostatin-1 was purchased from Abcam (Cambridge, UK). To detect autolysosomes, we employed the DALGreen agent (Dojindo Co., Kumamoto, Japan) according the manufacturers protocol. Briefly, DALGreen, which is a small hydrophobic molecule, passes the cell surface membrane of live cells and is Staurosporine biological activity incorporated in the autophagosome. After a lysosome fuses with the autophagosome, the incorporated DALGreen begins to fluoresce as the acidity increases [13], and this was visualized under a confocal fluorescence microscope (Leica TCS SP8; Leica Corporation, Germany) and analyzed with a Guava EasyCyte cell analyzer (Hayward, CA, USA). Cells were also stained with a fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide (PI) (PromoCell GmbH, Heidelberg, Germany) followed by analysis with a confocal fluorescence microscope and cell analyzer. Xenoplantation and CPI-613 treatment The experimental protocol was approved by the Animal Care Committee of Gifu Graduate School of Gifu, Japan (approval No. 27C80). SCID-beige (CB17.Cg-PrkdcscidLystbg-J/CrlCrlj) mice were purchased from Charles River Laboratories Japan Staurosporine biological activity (Sizuoka, Staurosporine biological activity Japan) and were housed in the Animal facility of the Gifu Graduate School of Gifu, Gifu, Japan. Mice were monitored for indicators of distress and were euthanized humanely according to the.