Table 1..
Primers used for real-time RT-PCR.
Table 2.
Primers used for making insert sequences.
Fig 1.
FA treatment upregulates the expression of fatty acid metabolism-related genes without ATP production in NRCMs.
NRCMs were treated with the indicated concentrations of the FA for 24 h. (A, D) Transcript expression was measured using real-time RT-PCR. (B, C, E) Amounts of acetyl-CoA, ATP, and β-hydroxybutyrate were quantified. Results are shown as the mean ± SEM (A, D: n = 9, B, C: n = 5, E: n = 3). *p < 0.05, **p < 0.01 vs. 0 μM FA by Dunnett test (A, D) and Student’s t-test (B, C, E).
Fig 2.
FA suppressed NRCM proliferation.
NRCMs were treated with the indicated concentrations of the FA for 24 h (A-F) or 48 h (G). (A-F) The proportion of Ki67, phospho-Histone H3 (pHH3), or Aurora B-positive NRCMs was analyzed using immunostaining. Cells were stained with anti-Ki67, pHH3, or Aurora B-antibodies (red). Cardiomyocytes and nuclei were labeled with anti-α-actinin antibody (green) and DAPI (blue), respectively. (A, C, E) Representative images. (B, D, F) Quantitative data. The bars indicate 100 μm. Arrowheads indicate Aurora B. (G) The amount of NRCMs was analyzed by cell counting assay using WST-8. Results are shown as the mean ± SEM (B: n = 9, D: n = 6, F: n = 3, G: n = 5). *p < 0.05, **p < 0.01 vs. 0 μM FA by Dunnett test (B) and Student’s t-test (D, F, G).
Fig 3.
δ upregulates fatty acid metabolism-related factors and suppresses NRCM proliferation. NRCMs were treated with fenofibrate (Feno), pioglitazone (Pio), or GW501516 (GW501) at 10 μM for 24 h. (A) Transcript expression was measured using real-time RT-PCR. (B, C) The proportion of Ki67-positive NRCMs was analyzed using immunostaining. Cells were stained with an anti-Ki67 antibody (red). Cardiomyocytes and nuclei were labeled with anti-α-actinin antibody (green) and DAPI (blue), respectively. (B) Representative images. (C) Quantitative data. The bars indicate 100 μm. Results are shown as the mean ± SEM (n = 6). *p < 0.05, **p < 0.01 by Tukey-Kramer test.
Fig 4.
δ represses the increase of fatty acid metabolism-related gene products and recovers the cell cycle arrest in response to FA treatment. NRCMs were pretreated with GW6471, T0070907 (T007), or GSK3787 (GSK) at 10 μM for 24 h, followed by the treatment of 500 μM FA for 24 h. (A) Transcript expression was measured using real-time RT-PCR. (B, C) The proportion of Ki67-positive NRCMs was analyzed using immunostaining. Cells were stained with an anti-Ki67 antibody (red). Cardiomyocytes and nuclei were labeled with anti-α-actinin antibody (green) and DAPI (blue), respectively. (B) Representative images. (C) Quantitative data. The bars indicate 100 μm. Results are shown as mean ± SEM. *p < 0.05, **p < 0.01 vs. FA (-), DMSO and #p < 0.05, ##p < 0.01 vs. FA (+), DMSO by Tukey-Kramer test.
Fig 5.
FA promoted NRCM maturation through PPAR
δ. NRCMs were treated with 500 μM FA (A) or 10 μM GW501516 (GW501) (B) for 48 h. Otherwise, NRCMs were pretreated with GSK3787 (GSK) at 10 μM for 24 h, followed by the treatment of 500 μM FA for 48 h (C). The expression of the transcripts was measured by real-time RT-PCR. Results are shown as the mean ± SEM (A: n = 5-6, B, C: n = 6). *p < 0.05, **p < 0.01 vs. 0 μM FA by Student’s t-test (A, B) and Tukey-Kramer test (C).
Fig 6.
The overexpression of PDK4 or HMGCS2 suppresses NRCM proliferation.
The NRCMs were infected with the indicated lentiviral vectors for 72 h. (A) Protein expression was measured using western blotting with anti-PDK4 and anti-HMGCS2 antibodies. Representative images are shown. (B, C) The proportion of Ki67-positive NRCMs was analyzed using immunostaining. Cells were stained with an anti-Ki67 antibody (red). Cardiomyocytes and nuclei were labeled with anti-α-actinin antibody (green) and DAPI (blue), respectively. (B) Representative images. (C) Quantitative data. The bars indicate 100 μm. Results are shown as mean ± SEM (n = 6). **p < 0.01 vs. LV-Luciferase by Dunnet test.
Fig 7.
The suppression of PDK4 or HMGCS2 abrogates FA-induced cell cycle arrest.
The NRCMs were infected with the indicated lentiviral vectors for 48 h, followed by the stimulation of 500 μM FA. (A, B) Protein expression was measured using western blotting with anti-PDK4 and anti-HMGCS2 antibodies. (C, D) The proportion of Ki67-positive NRCMs was analyzed using immunostaining. Cells were stained with an anti-Ki67 antibody (red). Cardiomyocytes and nuclei were labeled with anti-α-actinin antibody (green) and DAPI (blue), respectively. (A, C) Representative images. (B, D) Quantitative data. The bars indicate 100 μm. Results are shown as mean ± SEM (B: n = 4, D: n = 6). *p < 0.05, **p < 0.01 by Tukey-Kramer test.