Se transcription (RT)-PCR for MLC2v, MLC2a, a-MHC, ANF, Nkx2.5, GATA-4 and GAPDH was performed using standard procedures. Title Loaded From File Briefly, total RNA was prepared using Trizol reagent (Invitrogen). First strand cDNA was synthesized from 1 mg of total RNA, in a total volume of 20 mL, using oligo (dT)18 primer and a RevetAidTM First Strand cDNA Synthesis Kit. The RT-PCR was performed with GAPDH mRNA as a normalizing internal control. The resulting cDNA (50 ng) was amplified by PCR using specific primers. Primer sequences and PCR conditions are detailed in Table S1. Thermal cycling (in 20 mL) was performed as follows: 1531364 a 3 min denaturation at 94uC, 30 cycles of 94uC for 30 sec, 60uC for 30 sec and 72uC for 1 min, and a final extension for 6 min at 72uC. PCR Title Loaded From File products were resolved by electrophoresis on 1.5 agarose gels. They were visualized by UV transillumination and photographed. Semiquantitative analysis was done by Alphaview 1.3 software (Alpha Lnnotech Inc.).Real-Time PCRFor quantitative analysis on GATA-4, ANF, and a-MHC expressions, real-time PCR using above primers (detailed in Table S1) was performed as described previously [32]. Briefly, the processes of RNA extraction and reverse transcription of RNA (1 mg) were the same to Semi-quantitative RT-PCR. Real-time RT-PCR amplification reactions was performed in a final volume of 20 mL containing 50 ng cDNA, 10 mL of 26 iQSYBR-green mix (Takara, Japan), 300 nmol of forward and reverse primersAn Indirect Co-Culture Model for ESCsusing the LineGene 9660 real-time PCR Detection System (Bioer, China). The thermal cycling conditions comprised 95uC for 10 sec, 1 min at the corresponding annealing temperature, 53uC for 10 sec and 72uC for 40 sec. These settings were applied for 50 cycles. Specificity of amplification was determined by DNA melting curve during gradual temperature increments (0.5uC). The transcripts for GAPDH were used for internal normalization. Relative quantification was performed by the ggCT method.Confocal MicroscopyEB outgrowths were fixed in 4 paraformaldehyde for 30 min, permeabilized for 15 min with 0.25 Triton X-100, and blocked in 5 normal goat serum (NGS) for 15 min. Subsequently, cells were incubated with the primary antibody in a humidified chamber at 37uC for 2 h. Rabbit anti-cardiac troponin I (cTnI) antibody (Santa Cruz, CA) and anti-a-actinin antibody (sigma) were added at dilutions of 1:250 and 1:400, respectively. After washed with 0.4 Triton X-100 and PBS, cells were incubated at 37uC for 1662274 4 h to corresponding FITC-conjugated or Cy3-conjugated secondary antibodies at a dilution of 1:400. DAPI staining (Sigma, 1:1000) was used to identify nuclei. Analysis was performed using a confocal microscope (FV1000, Olympus).stained with annexin-V and 7-amino-actinomycin D (7-AAD) for 15 minutes according to the manufacturer’s instructions (BD Pharmingen). Within 1 hour after staining, cells were analyzed by flow cytometry using CellQuest software (Becton Dickinson). For cell proliferation assay, the samples were pulsed with 5-bromodeoxyuridine (BrdU) at 10 mmol/L for 18 hours before co-staining for BrdU and a-actinin. Rabbit anti-BrdU antibody (Santa Cruz, CA) and mouse anti-a-actinin antibody (sigma) were added at dilutions of 1:500 and 1:400, respectively. After washed with 0.4 Triton X-100 and PBS, cells were incubated at 37uC for 2 h to corresponding FITC-conjugated or Cy3-conjugated secondary antibodies at a dilution of 1:400. The cells were counterstained with DAPI (.Se transcription (RT)-PCR for MLC2v, MLC2a, a-MHC, ANF, Nkx2.5, GATA-4 and GAPDH was performed using standard procedures. Briefly, total RNA was prepared using Trizol reagent (Invitrogen). First strand cDNA was synthesized from 1 mg of total RNA, in a total volume of 20 mL, using oligo (dT)18 primer and a RevetAidTM First Strand cDNA Synthesis Kit. The RT-PCR was performed with GAPDH mRNA as a normalizing internal control. The resulting cDNA (50 ng) was amplified by PCR using specific primers. Primer sequences and PCR conditions are detailed in Table S1. Thermal cycling (in 20 mL) was performed as follows: 1531364 a 3 min denaturation at 94uC, 30 cycles of 94uC for 30 sec, 60uC for 30 sec and 72uC for 1 min, and a final extension for 6 min at 72uC. PCR products were resolved by electrophoresis on 1.5 agarose gels. They were visualized by UV transillumination and photographed. Semiquantitative analysis was done by Alphaview 1.3 software (Alpha Lnnotech Inc.).Real-Time PCRFor quantitative analysis on GATA-4, ANF, and a-MHC expressions, real-time PCR using above primers (detailed in Table S1) was performed as described previously [32]. Briefly, the processes of RNA extraction and reverse transcription of RNA (1 mg) were the same to Semi-quantitative RT-PCR. Real-time RT-PCR amplification reactions was performed in a final volume of 20 mL containing 50 ng cDNA, 10 mL of 26 iQSYBR-green mix (Takara, Japan), 300 nmol of forward and reverse primersAn Indirect Co-Culture Model for ESCsusing the LineGene 9660 real-time PCR Detection System (Bioer, China). The thermal cycling conditions comprised 95uC for 10 sec, 1 min at the corresponding annealing temperature, 53uC for 10 sec and 72uC for 40 sec. These settings were applied for 50 cycles. Specificity of amplification was determined by DNA melting curve during gradual temperature increments (0.5uC). The transcripts for GAPDH were used for internal normalization. Relative quantification was performed by the ggCT method.Confocal MicroscopyEB outgrowths were fixed in 4 paraformaldehyde for 30 min, permeabilized for 15 min with 0.25 Triton X-100, and blocked in 5 normal goat serum (NGS) for 15 min. Subsequently, cells were incubated with the primary antibody in a humidified chamber at 37uC for 2 h. Rabbit anti-cardiac troponin I (cTnI) antibody (Santa Cruz, CA) and anti-a-actinin antibody (sigma) were added at dilutions of 1:250 and 1:400, respectively. After washed with 0.4 Triton X-100 and PBS, cells were incubated at 37uC for 1662274 4 h to corresponding FITC-conjugated or Cy3-conjugated secondary antibodies at a dilution of 1:400. DAPI staining (Sigma, 1:1000) was used to identify nuclei. Analysis was performed using a confocal microscope (FV1000, Olympus).stained with annexin-V and 7-amino-actinomycin D (7-AAD) for 15 minutes according to the manufacturer’s instructions (BD Pharmingen). Within 1 hour after staining, cells were analyzed by flow cytometry using CellQuest software (Becton Dickinson). For cell proliferation assay, the samples were pulsed with 5-bromodeoxyuridine (BrdU) at 10 mmol/L for 18 hours before co-staining for BrdU and a-actinin. Rabbit anti-BrdU antibody (Santa Cruz, CA) and mouse anti-a-actinin antibody (sigma) were added at dilutions of 1:500 and 1:400, respectively. After washed with 0.4 Triton X-100 and PBS, cells were incubated at 37uC for 2 h to corresponding FITC-conjugated or Cy3-conjugated secondary antibodies at a dilution of 1:400. The cells were counterstained with DAPI (.
