First, the Hes3 gene itself is regulated by the treatments used here

ed with 2 ml of PKH26 at room temperature for 5 min. The labeled mixtures were dialyzed in 2 ml of 1% bovine serum albumin and ultracentrifuged 1692608 at 100,000 g for 60 min at 4uC to pellet the labeled MVs. After washed with EBM-2, the pellet was suspended with 1 ml of culture medium and added into H9c2 cells for 24 h incubation. The 49, 6-diamidino-2 -phenylindole was used for nuclear staining. Cell images were taken using an inverted microscope. Culture of EPCs The BM derived EPCs were cultured from adult C57BL/6J genetic background mice as we previously described. Mouse tibias and femurs were taken under deep anesthesia and BM was flushed out from tibias and femurs. BM mononuclear cells were isolated by using density gradient centrifuge method. After being washed with Phoshate-buffered saline, BM MNCs were counted and plated on a 25 cm2 flask then grown in endothelial cell basal medium supplemented with 5% FBS containing EPC growth cytokine cocktail. After 3 days in culture, non-adherent cells were removed by washing with PBS. Thereafter, culture medium was changed every 2 days. Measurement of Cell Surface Area The surface area of CMs in different groups was measured according to the method of Simpson. In brief, cell images were captured by a 206magnification digital inverted microscope. Then the images of CMs were traced and the cell surface areas were analyzed by using Image J software. The surface areas of CMs in 6 different fields were averaged. The surface area data in each treatment group was presented as the rate of that in control group. Preparation of EPC-MVs and RNA-free EPC-MVs After being cultured for 7 d, EPC cultures were washed with PBS, and incubated with serum-free medium overnight. The conditional medium which contained EPC secretions was collected and centrifuged at 4uC. Then the supernatant was ultracentrifuged at 4uC to pellet EPCMVs. For preparation of RNA-free EPC-MVs, we disrupted the EPC-MVs with ribonuclease A . First, the EPC-MVs were incubated with 0.1% Triton X-100 for 5 min. Then the MV fraction was added in 200 U/ml of RNase A for 90 min at 37uC. After that, EPC-MVs were ultracentrifuged at 4uC to pellet the RNA deleted MVs for the following experiments. To verify the effect of RNase A on MVs, the total RNAs were isolated from EPC-MVs and EPC-rdMVs using the RNA Cobicistat price Isolation Kit Methyl Thiazolyl Tetrazolium Assay The viabilities of H9c2 CMs after different treatments were 6099352 determined using the MTT Assay Kit by following the manufacture’s protocol. The CMs culture was replaced with 100 ml of fresh culture medium. Cells in 96-well plate were added in 10 ml of 12 mM MTT solution and incubated at 37uC for 4 h. Then 100 ml of the sodium dodecyl sulfate HCl solution was added to each well and incubated at 37uC for 4 h. Finally, the 96-well plate was read by a microtiterplate reader at 535 nm. The percentage of viability was defined as the relative absorbance of the treated cells versus the untreated controls. Protective Effects of EPC-MVs on Cardiomyocytes Flow Cytometry Analysis of Cell Apoptosis The CM apoptosis was assessed by using an Apoptosis Assay Kit. The H9c2 CMs were collected by using 0.25% trypsin, and centrifuged at 200 g for 7 min. Cells were resuspended in 100 ml annexin-binding buffer, and incubated with 5 ml of annexin V-FITC and 1 ml of propidium iodide at RT in the dark for 15 min. Apoptotic cells were detected by a flow cytometer. The CMs stained with both annexin V and PI were considered to be lated with 2 ml of PKH26 at room temperature for 5 min. The labeled mixtures were dialyzed in 2 ml of 1% bovine serum albumin and ultracentrifuged at 100,000 g for 60 min at 4uC to pellet the labeled MVs. After washed with EBM-2, the pellet was suspended with 1 ml of culture medium and added into H9c2 cells for 24 h incubation. The 49, 6-diamidino-2 -phenylindole was used for nuclear staining. Cell images were taken using an inverted microscope. Culture of EPCs The BM derived EPCs were cultured from adult C57BL/6J genetic background mice as we previously described. Mouse tibias and femurs were taken under deep anesthesia and BM was flushed out from tibias and femurs. BM mononuclear cells were isolated by 18083779 using density gradient centrifuge method. After being washed with Phoshate-buffered saline, BM MNCs were counted and plated on a 25 cm2 flask then grown in endothelial cell basal medium supplemented with 5% FBS containing EPC growth cytokine cocktail. After 3 days in culture, non-adherent cells were removed by washing with PBS. Thereafter, culture medium was changed every 2 days. Measurement of Cell Surface Area The surface area of CMs in different groups was measured according to the method of Simpson. In brief, cell images were captured by a 206magnification digital inverted microscope. Then the images of CMs were traced and the cell surface areas were analyzed by using Image J software. The surface areas of CMs in 6 different fields were averaged. The surface area data in each treatment group was presented as the rate of that in control group. Preparation of EPC-MVs and RNA-free EPC-MVs After being cultured for 7 d, EPC cultures were washed with PBS, and incubated with serum-free medium overnight. The conditional medium which contained EPC secretions was collected and centrifuged at 4uC. Then the supernatant was ultracentrifuged at 4uC to pellet EPCMVs. For preparation of RNA-free EPC-MVs, we disrupted the EPC-MVs with ribonuclease A . First, the EPC-MVs were incubated with 0.1% Triton X-100 for 5 min. Then the MV fraction was added in 200 U/ml of RNase A for 90 min at 37uC. After that, EPC-MVs were ultracentrifuged at 4uC to pellet the RNA deleted MVs for the following experiments. To verify the effect of RNase A on MVs, the total RNAs were isolated from EPC-MVs and EPC-rdMVs using the RNA Isolation Kit Methyl Thiazolyl Tetrazolium Assay The viabilities of H9c2 CMs after different treatments were determined using the MTT Assay Kit by following the manufacture’s protocol. The CMs culture was replaced with 100 ml of fresh culture medium. Cells in 96-well plate were added in 10 ml of 12 mM MTT solution and incubated at 37uC for 4 h. Then 100 ml of the sodium dodecyl sulfate HCl solution was added to each well and incubated at 37uC for 4 h. Finally, the 96-well plate was read by a microtiterplate reader at 535 nm. The percentage of viability was defined as the relative absorbance of the treated cells versus the untreated controls. Protective Effects of EPC-MVs on Cardiomyocytes Flow Cytometry Analysis of Cell Apoptosis The CM apoptosis was assessed by using an Apoptosis Assay Kit. The H9c2 CMs were collected by using 0.25% trypsin, and centrifuged at 200 g for 7 min. 1417961 Cells were resuspended in 100 ml annexin-binding buffer, and incubated with 5 ml of annexin V-FITC and 1 ml of propidium iodide at RT in the dark for 15 min. Apoptotic cells were detected by a flow cytometer. The CMs stained with both annexin V and PI were considered to be lat