Journal: Oxidative Medicine and Cellular Longevity

Article Title: Activation of the M3AChR and Notch1/HSF1 Signaling Pathway by Choline Alleviates Angiotensin II-Induced Cardiomyocyte Apoptosis

doi: 10.1155/2021/9979706

Figure Lengend Snippet: Choline attenuated angiotensin II- (Ang II) induced stimulation of the mitochondrial death pathway and cardiomyocyte apoptosis. (a) 4-Diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) administration blocked the effect of choline on terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling- (TUNEL-) positive neonatal rat ventricular myocytes (NRVMs). Scale bar: 50 μ m. (b) The ratio of TUNEL-positive cells to total cells. Data are presented as means ± SD ( n = 5 independent experiments). (c) Choline treatment increased the Bcl-2/Bax ratio and inhibited caspase-3 activation. The protective effects of choline were reversed by 4-DAMP. (d, e) Quantification of Bcl-2/Bax and cleaved-caspase-3. Data are presented as means ± SD ( n = 5 independent experiments). (f) Representative image and graph showing levels of Omi/high-temperature requirement protein A2 (HtrA2), the second mitochondrial activator of caspases/direct inhibitor of apoptosis-binding protein with low P (Smac/DIABLO), and cytochrome c in the cytosol fraction. Data are presented as means ± SD ( n = 5 independent experiments). (g) Representative western blot images and data of Omi/HtrA2, Smac/DIABLO, and cytochrome c in the mitochondrial fraction. Data are presented as means ± SD ( n = 5 independent experiments). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 versus control (CTR); # P < 0.05, ## P < 0.01, and ### P < 0.001 versus Ang II; & P < 0.05, && P < 0.01, and &&& P < 0.001 versus the choline-treated Ang II group.

Article Snippet: The primary antibodies used in the experiments were as follows: Bcl-2 (1 : 2000; Cell Signaling Technology), Bax (1 : 1000; Cell Signaling Technology), Omi/HtrA2 (1 : 1000; Cell Signaling Technology), Smac/DIABLO (1 : 1000; Cell Signaling Technology), cytochrome c (1 : 1000; Cell Signaling Technology), Notch1 intracellular domain (NICD, 1 : 2000; ThermoFisher Scientific), HSF1 (1 : 1000; Thermo Fisher Scientific), and cleaved-caspase-3 (1 : 1000; Cell Signaling Technology).

Techniques: End Labeling, TUNEL Assay, Activation Assay, Binding Assay, Western Blot

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Activation of the M3AChR and Notch1/HSF1 Signaling Pathway by Choline Alleviates Angiotensin II-Induced Cardiomyocyte Apoptosis

doi: 10.1155/2021/9979706

Figure Lengend Snippet: Downregulation of type 3 muscarinic acetylcholine receptors (M3AChR) blocked the protective effect of choline on Ang II-induced cardiomyocyte apoptosis. (a) Choline increased the Bcl-2/Bax ratio and inhibited cleaved-caspase-3 expression; these effects were abolished by M3AChR small interfering ribonucleic acids (siRNAs). (b, c) Quantification of Bcl-2/Bax and cleaved-caspase-3. Data are presented as means ± SD ( n = 5 independent experiments). (d) Representative image and graph of Omi/HtrA2, Smac/DIABLO, and cytochrome c protein levels in the cytosol fraction. Data are presented as means ± SD ( n = 5 independent experiments). (e) Representative western blot images and data of Omi/HtrA2, Smac/DIABLO, and cytochrome c levels in the mitochondrial fraction. Data are presented as means ± SD ( n = 5 independent experiments). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 vs. the negative control (NC) siRNA group; # P < 0.05, ## P < 0.01, and ### P < 0.001 versus the choline-treated NC siRNA group.

Article Snippet: The primary antibodies used in the experiments were as follows: Bcl-2 (1 : 2000; Cell Signaling Technology), Bax (1 : 1000; Cell Signaling Technology), Omi/HtrA2 (1 : 1000; Cell Signaling Technology), Smac/DIABLO (1 : 1000; Cell Signaling Technology), cytochrome c (1 : 1000; Cell Signaling Technology), Notch1 intracellular domain (NICD, 1 : 2000; ThermoFisher Scientific), HSF1 (1 : 1000; Thermo Fisher Scientific), and cleaved-caspase-3 (1 : 1000; Cell Signaling Technology).

Techniques: Expressing, Western Blot, Negative Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Activation of the M3AChR and Notch1/HSF1 Signaling Pathway by Choline Alleviates Angiotensin II-Induced Cardiomyocyte Apoptosis

doi: 10.1155/2021/9979706

Figure Lengend Snippet: Downregulation of Notch1 blocked the protective effect of choline on Ang II-induced apoptosis. (a) Representative image and graph of Bcl-2, Bax, and cleaved-caspase-3 protein levels. (b, c) Quantification of Bcl-2/Bax and cleaved-caspase-3. Data are presented as means ± SD ( n = 5 independent experiments). (d) Representative image and graph of Omi/HtrA2, Smac/DIABLO, and cytochrome c protein levels in the cytosol fraction. Data are presented as means ± SD ( n = 5 independent experiments). (e) Representative western blot images and data of Omi/HtrA2, Smac/DIABLO, and cytochrome c protein levels in the mitochondrial fraction. Data are presented as means ± SD ( n = 5 independent experiments). ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001 versus the NC siRNA group; # P < 0.05, ## P < 0.01, and ### P < 0.001 versus the choline-treated negative control (NC) siRNA group.

Article Snippet: The primary antibodies used in the experiments were as follows: Bcl-2 (1 : 2000; Cell Signaling Technology), Bax (1 : 1000; Cell Signaling Technology), Omi/HtrA2 (1 : 1000; Cell Signaling Technology), Smac/DIABLO (1 : 1000; Cell Signaling Technology), cytochrome c (1 : 1000; Cell Signaling Technology), Notch1 intracellular domain (NICD, 1 : 2000; ThermoFisher Scientific), HSF1 (1 : 1000; Thermo Fisher Scientific), and cleaved-caspase-3 (1 : 1000; Cell Signaling Technology).

Techniques: Western Blot, Negative Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Activation of the M3AChR and Notch1/HSF1 Signaling Pathway by Choline Alleviates Angiotensin II-Induced Cardiomyocyte Apoptosis

doi: 10.1155/2021/9979706

Figure Lengend Snippet: Choline likely attenuates Ang II-induced cardiomyocyte apoptosis by inhibiting mtROS generation and the release of proapoptotic proteins from the mitochondria via the M3AChR and Notch1/HSF1 pathway. Ang II: angiotensin II; M3AChR: type 3 muscarinic acetylcholine receptor; HSF1: heat shock transcription factor 1; Omi/HtrA2: Omi/high-temperature requirement protein A2; Smac/DIABLO: second mitochondrial activator of caspases/direct inhibitor of apoptosis-binding protein with low P; mtROS: mitochondrial reactive oxygen species.

Article Snippet: The primary antibodies used in the experiments were as follows: Bcl-2 (1 : 2000; Cell Signaling Technology), Bax (1 : 1000; Cell Signaling Technology), Omi/HtrA2 (1 : 1000; Cell Signaling Technology), Smac/DIABLO (1 : 1000; Cell Signaling Technology), cytochrome c (1 : 1000; Cell Signaling Technology), Notch1 intracellular domain (NICD, 1 : 2000; ThermoFisher Scientific), HSF1 (1 : 1000; Thermo Fisher Scientific), and cleaved-caspase-3 (1 : 1000; Cell Signaling Technology).

Techniques: Binding Assay