Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: Acetylcholine attenuates hypoxia/ reoxygenation-induced mitochondrial and cytosolic ROS formation in H9c2 cells via M2 acetylcholine receptor.

doi: 10.1159/000343360

Figure Lengend Snippet: Fig. 5. Role of M2 AChR on the levels of ROS in the presence of ACh during H/R. (A) The silencing efficiency of M2 AChR siRNA were detected by Western blotting, equal protein loading was confirmed with the GAPDH antibody. (B) The effects of knockdown of M2 AChR on cell injury were assayed by MTT. (C, D) M2 AChR small siRNA transfection increases the contents of ROS in the presence of ACh during H/R. All the transfection groups were exposed to H/R with ACh followed by DCFH-DA staining 48 h after transfection. Data are presented as means ± SEM. n = 4. **P < 0.01 vs NC siRNA group; ##P < 0.01 vs NC siRNA exposed to HR with ACh group. Scale bar = 50 µm.

Article Snippet: Gels were then analyzed by Western blot with the following antibodies: GAPDH Mouse Monoclonal Antibody (Sinopept), mAChR M2 pAb, caspase-3 pAb (Bioworld Technology), rac 1 pAb (Cell Signaling Technology).

Techniques: Western Blot, Knockdown, Transfection, Staining

Journal: Molecular medicine reports

Article Title: Synergistic effects of valproic acid and arsenic trioxide on RPMI8226 cells in vitro and the possible underlying mechanisms.

doi: 10.3892/mmr.2015.3519

Figure Lengend Snippet: Figure 4. mRNA expression levels of Bax, Bcl‑2, Caspase 8, Caspase 9, HDAC1 and LSD1 in the RPMI8226 cells: 1, ABcl‑2/β‑actin; 2, ABax/ β‑actin; 3, ACaspase 8/β‑actin; 4, ACaspase 9/β‑actin; 5, AHDAC1/β‑actin; 6, ALSD1/β‑actin. *P<0.05, vs. control. Data are expressed as the mean ± stan dard deviation VPA, valproic acid; ATO, arsenic trioxide.

Article Snippet: The rabbit anti-human monoclonal Bcl-2, Bax, Caspase 8, Caspase 9, LSD1 and JMJD2B antibodies, and rabbit anti-human polyclonal acetylated-H3 and H3K9me2 antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA), and mouse anti-human monoclonal β-actin antobidy was purchased from Beijing Sinopept Biotechnology, Co., Ltd. (Beijing, China).

Techniques: Expressing, Control

Journal: Molecular medicine reports

Article Title: Synergistic effects of valproic acid and arsenic trioxide on RPMI8226 cells in vitro and the possible underlying mechanisms.

doi: 10.3892/mmr.2015.3519

Figure Lengend Snippet: Figure 5. Protein expression levels of Bcl‑2, Bax, Caspase 8, Caspase 9, acetylated H3, LSD1 and H3K9me2 in the RPMI8226 cells, analyzed by western blot analysis. 1, control group; 2, VPA 1 mmol/l; 3, VPA 5 mmol/l; 4, ATO 4 µmol/l; 5, VPA 1 mmol/l+ATO 4 µmol/l; 6, VPA 5 mmol/l+ATO 4 µmol/l. VPA, valproic acid; ATO, arsenic trioxide.

Article Snippet: The rabbit anti-human monoclonal Bcl-2, Bax, Caspase 8, Caspase 9, LSD1 and JMJD2B antibodies, and rabbit anti-human polyclonal acetylated-H3 and H3K9me2 antibodies were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA), and mouse anti-human monoclonal β-actin antobidy was purchased from Beijing Sinopept Biotechnology, Co., Ltd. (Beijing, China).

Techniques: Expressing, Western Blot, Control

Journal: Journal of cellular physiology

Article Title: Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through PINK1/Parkin Signal Pathway in H9c2 Cells.

doi: 10.1002/jcp.25215

Figure Lengend Snippet: Fig. 1. H/R caused cell injury and defective mitophagy during different reoxygenation periods. (A) H/R reduced cell viability. (B and C) H/R increased the level of cleaved-caspase 3. (D–H) H/R led to impaired autophagy and mitophagy, appearing as decreased levels of LC3-II/LC3-I, accumulation of p62, and suppressive mitochondrial translocation of PINK1 and Parkin by western blot analysis. P < 0.05 and P < 0.01 versus control group. (n ¼ 6).

Article Snippet: After blocking, blots were probed with primary antibodies against LC3 (1:1000 dilution, CST, USA, 4108), SQSTM1/p62 (1:1000 dilution, CST, USA, 5114), PINK1 (1:2000 dilution, Abcam, USA, Ab23707), Parkin (1:1000 dilution, Abcam, USA, Ab77924), caspase 3 (1:1000 dilution, CST, USA, 9662) cleaved-caspase 3 (1:1000 dilution, CST, USA, 9661), cytochrome c (1:1000 dilution, CST, USA, 4272S), GAPDH (1:5000 dilution, Sinopept, China, 41301M), and Subunit IV of cytochrome oxidase (COX IV, 1:1000 dilution, CST, USA, 4850) overnight at 4°C, followed by horseradish peroxidaseconjugated secondary antibody for 30min at 37°C.

Techniques: Translocation Assay, Western Blot, Control

Journal: Journal of cellular physiology

Article Title: Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through PINK1/Parkin Signal Pathway in H9c2 Cells.

doi: 10.1002/jcp.25215

Figure Lengend Snippet: Fig. 2. ACh induced cytoprotective mitophagy following H/R. (A) ACh (10-8–10-5–M) increased cell viability in concentration-dependent manner. (B and C) ACh reduced the level of cleaved-caspase 3. (D–H) ACh promoted autophagy and mitophagy, appearing as increased levels of LC3-II/LC3-I, decreased p62, and mitochondrial translocation of PINK1 and Parkin by western blot analysis. P < 0.05 and P < 0.01 versus control group. #P < 0.05 and ##P < 0.01 versus H/R group. (n ¼ 6).

Article Snippet: After blocking, blots were probed with primary antibodies against LC3 (1:1000 dilution, CST, USA, 4108), SQSTM1/p62 (1:1000 dilution, CST, USA, 5114), PINK1 (1:2000 dilution, Abcam, USA, Ab23707), Parkin (1:1000 dilution, Abcam, USA, Ab77924), caspase 3 (1:1000 dilution, CST, USA, 9662) cleaved-caspase 3 (1:1000 dilution, CST, USA, 9661), cytochrome c (1:1000 dilution, CST, USA, 4272S), GAPDH (1:5000 dilution, Sinopept, China, 41301M), and Subunit IV of cytochrome oxidase (COX IV, 1:1000 dilution, CST, USA, 4850) overnight at 4°C, followed by horseradish peroxidaseconjugated secondary antibody for 30min at 37°C.

Techniques: Concentration Assay, Translocation Assay, Western Blot, Control

Journal: Journal of cellular physiology

Article Title: Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through PINK1/Parkin Signal Pathway in H9c2 Cells.

doi: 10.1002/jcp.25215

Figure Lengend Snippet: Fig. 3. M2 receptor antagonist METH suppressed ACh-activated mitophagy. (A–E) Expressions of autophagy/mitophagy markers including LC3, p62, PINK1, and Parkin. (F and G) Co-localization of Parkin and MitoTracker Green-labeled mitochondria. Fluorescent images were captured by confocal microscopy and columns represent the Manders overlap coefficient of Parkin and mitochondria. At least 62 cells from three independent experiments for each group were included. Scale bar: 50 mm. P < 0.05 and P < 0.01 versus control group. #P < 0.05 versus H/R group. &P < 0.05 versus H/R þ ACh group.

Article Snippet: After blocking, blots were probed with primary antibodies against LC3 (1:1000 dilution, CST, USA, 4108), SQSTM1/p62 (1:1000 dilution, CST, USA, 5114), PINK1 (1:2000 dilution, Abcam, USA, Ab23707), Parkin (1:1000 dilution, Abcam, USA, Ab77924), caspase 3 (1:1000 dilution, CST, USA, 9662) cleaved-caspase 3 (1:1000 dilution, CST, USA, 9661), cytochrome c (1:1000 dilution, CST, USA, 4272S), GAPDH (1:5000 dilution, Sinopept, China, 41301M), and Subunit IV of cytochrome oxidase (COX IV, 1:1000 dilution, CST, USA, 4850) overnight at 4°C, followed by horseradish peroxidaseconjugated secondary antibody for 30min at 37°C.

Techniques: Labeling, Confocal Microscopy, Control

Journal: Journal of cellular physiology

Article Title: Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through PINK1/Parkin Signal Pathway in H9c2 Cells.

doi: 10.1002/jcp.25215

Figure Lengend Snippet: Fig. 4. M2 receptor siRNA attenuated ACh-mediated mitophagy. (A–E) Expressions of autophagy/mitophagy markers including LC3, p62, PINK1, and Parkin after M2 receptor siRNA transfection. (F and G) Co-localization of Parkin and MitoTracker Green-labeled mitochondria. Fluorescent images were captured by confocal microscopy and columns represent the Manders overlap coefficient of Parkin and mitochondria. At least 78 cells from three independent experiments for each groupwere included. Scale bar: 50 mm. P < 0.05 and P < 0.01 versus control group. #P < 0.05 versus H/R group.

Article Snippet: After blocking, blots were probed with primary antibodies against LC3 (1:1000 dilution, CST, USA, 4108), SQSTM1/p62 (1:1000 dilution, CST, USA, 5114), PINK1 (1:2000 dilution, Abcam, USA, Ab23707), Parkin (1:1000 dilution, Abcam, USA, Ab77924), caspase 3 (1:1000 dilution, CST, USA, 9662) cleaved-caspase 3 (1:1000 dilution, CST, USA, 9661), cytochrome c (1:1000 dilution, CST, USA, 4272S), GAPDH (1:5000 dilution, Sinopept, China, 41301M), and Subunit IV of cytochrome oxidase (COX IV, 1:1000 dilution, CST, USA, 4850) overnight at 4°C, followed by horseradish peroxidaseconjugated secondary antibody for 30min at 37°C.

Techniques: Transfection, Labeling, Confocal Microscopy, Control

Journal: Journal of cellular physiology

Article Title: Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through PINK1/Parkin Signal Pathway in H9c2 Cells.

doi: 10.1002/jcp.25215

Figure Lengend Snippet: Fig. 1. H/R caused cell injury and defective mitophagy during different reoxygenation periods. (A) H/R reduced cell viability. (B and C) H/R increased the level of cleaved-caspase 3. (D–H) H/R led to impaired autophagy and mitophagy, appearing as decreased levels of LC3-II/LC3-I, accumulation of p62, and suppressive mitochondrial translocation of PINK1 and Parkin by western blot analysis. P < 0.05 and P < 0.01 versus control group. (n ¼ 6).

Article Snippet: After blocking, blots were probed with primary antibodies against LC3 (1:1000 dilution, CST, USA, 4108), SQSTM1/p62 (1:1000 dilution, CST, USA, 5114), PINK1 (1:2000 dilution, Abcam, USA, Ab23707), Parkin (1:1000 dilution, Abcam, USA, Ab77924), caspase 3 (1:1000 dilution, CST, USA, 9662) cleaved-caspase 3 (1:1000 dilution, CST, USA, 9661), cytochrome c (1:1000 dilution, CST, USA, 4272S), GAPDH (1:5000 dilution, Sinopept, China, 41301M), and Subunit IV of cytochrome oxidase (COX IV, 1:1000 dilution, CST, USA, 4850) overnight at 4°C, followed by horseradish peroxidaseconjugated secondary antibody for 30min at 37°C.

Techniques: Translocation Assay, Western Blot, Control

Journal: Journal of cellular physiology

Article Title: Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through PINK1/Parkin Signal Pathway in H9c2 Cells.

doi: 10.1002/jcp.25215

Figure Lengend Snippet: Fig. 2. ACh induced cytoprotective mitophagy following H/R. (A) ACh (10-8–10-5–M) increased cell viability in concentration-dependent manner. (B and C) ACh reduced the level of cleaved-caspase 3. (D–H) ACh promoted autophagy and mitophagy, appearing as increased levels of LC3-II/LC3-I, decreased p62, and mitochondrial translocation of PINK1 and Parkin by western blot analysis. P < 0.05 and P < 0.01 versus control group. #P < 0.05 and ##P < 0.01 versus H/R group. (n ¼ 6).

Article Snippet: After blocking, blots were probed with primary antibodies against LC3 (1:1000 dilution, CST, USA, 4108), SQSTM1/p62 (1:1000 dilution, CST, USA, 5114), PINK1 (1:2000 dilution, Abcam, USA, Ab23707), Parkin (1:1000 dilution, Abcam, USA, Ab77924), caspase 3 (1:1000 dilution, CST, USA, 9662) cleaved-caspase 3 (1:1000 dilution, CST, USA, 9661), cytochrome c (1:1000 dilution, CST, USA, 4272S), GAPDH (1:5000 dilution, Sinopept, China, 41301M), and Subunit IV of cytochrome oxidase (COX IV, 1:1000 dilution, CST, USA, 4850) overnight at 4°C, followed by horseradish peroxidaseconjugated secondary antibody for 30min at 37°C.

Techniques: Concentration Assay, Translocation Assay, Western Blot, Control

Journal: Journal of cellular physiology

Article Title: Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through PINK1/Parkin Signal Pathway in H9c2 Cells.

doi: 10.1002/jcp.25215

Figure Lengend Snippet: Fig. 3. M2 receptor antagonist METH suppressed ACh-activated mitophagy. (A–E) Expressions of autophagy/mitophagy markers including LC3, p62, PINK1, and Parkin. (F and G) Co-localization of Parkin and MitoTracker Green-labeled mitochondria. Fluorescent images were captured by confocal microscopy and columns represent the Manders overlap coefficient of Parkin and mitochondria. At least 62 cells from three independent experiments for each group were included. Scale bar: 50 mm. P < 0.05 and P < 0.01 versus control group. #P < 0.05 versus H/R group. &P < 0.05 versus H/R þ ACh group.

Article Snippet: After blocking, blots were probed with primary antibodies against LC3 (1:1000 dilution, CST, USA, 4108), SQSTM1/p62 (1:1000 dilution, CST, USA, 5114), PINK1 (1:2000 dilution, Abcam, USA, Ab23707), Parkin (1:1000 dilution, Abcam, USA, Ab77924), caspase 3 (1:1000 dilution, CST, USA, 9662) cleaved-caspase 3 (1:1000 dilution, CST, USA, 9661), cytochrome c (1:1000 dilution, CST, USA, 4272S), GAPDH (1:5000 dilution, Sinopept, China, 41301M), and Subunit IV of cytochrome oxidase (COX IV, 1:1000 dilution, CST, USA, 4850) overnight at 4°C, followed by horseradish peroxidaseconjugated secondary antibody for 30min at 37°C.

Techniques: Labeling, Confocal Microscopy, Control

Journal: Journal of cellular physiology

Article Title: Acetylcholine Attenuates Hypoxia/Reoxygenation Injury by Inducing Mitophagy Through PINK1/Parkin Signal Pathway in H9c2 Cells.

doi: 10.1002/jcp.25215

Figure Lengend Snippet: Fig. 4. M2 receptor siRNA attenuated ACh-mediated mitophagy. (A–E) Expressions of autophagy/mitophagy markers including LC3, p62, PINK1, and Parkin after M2 receptor siRNA transfection. (F and G) Co-localization of Parkin and MitoTracker Green-labeled mitochondria. Fluorescent images were captured by confocal microscopy and columns represent the Manders overlap coefficient of Parkin and mitochondria. At least 78 cells from three independent experiments for each groupwere included. Scale bar: 50 mm. P < 0.05 and P < 0.01 versus control group. #P < 0.05 versus H/R group.

Article Snippet: After blocking, blots were probed with primary antibodies against LC3 (1:1000 dilution, CST, USA, 4108), SQSTM1/p62 (1:1000 dilution, CST, USA, 5114), PINK1 (1:2000 dilution, Abcam, USA, Ab23707), Parkin (1:1000 dilution, Abcam, USA, Ab77924), caspase 3 (1:1000 dilution, CST, USA, 9662) cleaved-caspase 3 (1:1000 dilution, CST, USA, 9661), cytochrome c (1:1000 dilution, CST, USA, 4272S), GAPDH (1:5000 dilution, Sinopept, China, 41301M), and Subunit IV of cytochrome oxidase (COX IV, 1:1000 dilution, CST, USA, 4850) overnight at 4°C, followed by horseradish peroxidaseconjugated secondary antibody for 30min at 37°C.

Techniques: Transfection, Labeling, Confocal Microscopy, Control