Journal: Cardiovascular Toxicology

Article Title: Overexpression of Programmed Cell Death 1 Prevents Doxorubicin-Induced Apoptosis Through Autophagy Induction in H9c2 Cardiomyocytes

doi: 10.1007/s12012-022-09726-w

Figure Lengend Snippet: Detection of Pdcd1 expression and autophagy in Pdcd1-overexpressing H9c2 (H9c2/Pdcd1) cells and negative control (H9c2/mock) cells. Pdcd1-overexpressing cells were established by transfecting H9c2 cells with Pdcd1-encoding plasmid DNA. After 24 h of transfection, the Pdcd1 protein expression (red fluorescence) was determined through immunofluorescence analysis in H9c2/mock ( a ) or H9c2/Pdcd1 cells ( b ). Autophagy was detected through staining with DAL Green, an autolysosome detection reagent (green fluorescence), and by the expression of LC3B, an autophagy marker, using immunofluorescence analysis in H9c2/mock ( c , e ) and H9c2/Pdcd1 cells ( d , f ). Magnification = × 400. Scale bar = 100 μm

Article Snippet: Primary antibody against Pdcd1 was purchased from Proteintech (Rosemont, IL, USA).

Techniques: Expressing, Negative Control, Plasmid Preparation, Transfection, Fluorescence, Immunofluorescence, Staining, Marker

Journal: Cardiovascular Toxicology

Article Title: Overexpression of Programmed Cell Death 1 Prevents Doxorubicin-Induced Apoptosis Through Autophagy Induction in H9c2 Cardiomyocytes

doi: 10.1007/s12012-022-09726-w

Figure Lengend Snippet: Effects of Pdcd1 overexpression on doxorubicin (DOX)-induced apoptosis and caspase-3/7 activation in H9c2 cells. a After the cells were incubated with 1 μM DOX for various time periods (1–24 h), apoptosis was evaluated through a luciferase assay indicating the increase in Annexin V-positive cells. b Activity of caspase-3/7, an executor enzyme of apoptosis, was assessed using a luciferase assay in cells incubated with 1 μM DOX for 8 h; the values were expressed as the percentage of DOX-untreated H9c2/mock control cells. c Apoptosis levels in response to various concentrations of DOX (0.03–1 μM). The apoptosis was determined after a 24 h incubation with DOX, using the Annexin V luciferase assay. Each value was expressed as a percentage of that in H9c2/mock control cells. d Nuclear morphological observations and quantification of the apoptotic cells stained with H33342 after a 24 h incubation with various concentrations of DOX (0.1–1 μM). The yellow arrows indicate a typical feature of apoptotic cells. Magnification = × 200. Scale bar = 50 μm. Data are presented as the mean ± SEM of three samples. * p < 0.05, ** p < 0.01 vs. the DOX-untreated mock (control) cells. ## p < 0.01 vs. the DOX-untreated Pdcd1 over-expressed cells. † p < 0.05, †† p < 0.01 vs. the corresponding DOX-treated mock cells

Article Snippet: Primary antibody against Pdcd1 was purchased from Proteintech (Rosemont, IL, USA).

Techniques: Over Expression, Activation Assay, Incubation, Luciferase, Activity Assay, Control, Staining

Journal: Cardiovascular Toxicology

Article Title: Overexpression of Programmed Cell Death 1 Prevents Doxorubicin-Induced Apoptosis Through Autophagy Induction in H9c2 Cardiomyocytes

doi: 10.1007/s12012-022-09726-w

Figure Lengend Snippet: Expression of phosphorylated mTOR (p-mTOR) and mTOR proteins in H9c2/mock and H9c2/Pdcd1 cells under DOX-untreated basal condition. a The expression of p-mTOR and mTOR in both cell types were evaluated through western blotting. b Quantitative levels of p-mTOR/mTOR protein expression. Data are presented as the mean ± SEM of three samples. * p < 0.05 vs. the mock cells. c Immunofluorescence detection of p-mTOR expression in H9c2/mock and H9c2/Pdcd1 cells. Magnification = × 200. Scale bar = 50 μm

Article Snippet: Primary antibody against Pdcd1 was purchased from Proteintech (Rosemont, IL, USA).

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: Cardiovascular Toxicology

Article Title: Overexpression of Programmed Cell Death 1 Prevents Doxorubicin-Induced Apoptosis Through Autophagy Induction in H9c2 Cardiomyocytes

doi: 10.1007/s12012-022-09726-w

Figure Lengend Snippet: Expression of Beclin-1, Atg5, and Atg3 proteins in H9c2/mock and H9c2/Pdcd1 cells under DOX-untreated basal condition. a The expression of Beclin-1, Atg5, and Atg3 in both cell types were evaluated using western blotting, with β-actin as the loading control. b – d Quantitative levels of protein expression. Data are presented as the mean ± SEM of three samples. * p < 0.05 vs. the mock cells

Article Snippet: Primary antibody against Pdcd1 was purchased from Proteintech (Rosemont, IL, USA).

Techniques: Expressing, Western Blot, Control

Journal: Cardiovascular Toxicology

Article Title: Overexpression of Programmed Cell Death 1 Prevents Doxorubicin-Induced Apoptosis Through Autophagy Induction in H9c2 Cardiomyocytes

doi: 10.1007/s12012-022-09726-w

Figure Lengend Snippet: Expression levels of caspase-3, Bad, and Bax proteins in H9c2/mock and H9c2/Pdcd1 cells under DOX-untreated basal condition. a The expression of the caspase-3, Bad, and Bax proteins in both cell types were evaluated using western blotting, with β-actin as the loading control. b – d Quantitative levels of protein expression. Data are presented as the mean ± SEM of three samples

Article Snippet: Primary antibody against Pdcd1 was purchased from Proteintech (Rosemont, IL, USA).

Techniques: Expressing, Western Blot, Control

Journal: Cardiovascular Toxicology

Article Title: Overexpression of Programmed Cell Death 1 Prevents Doxorubicin-Induced Apoptosis Through Autophagy Induction in H9c2 Cardiomyocytes

doi: 10.1007/s12012-022-09726-w

Figure Lengend Snippet: Changes in the basal levels of autophagy and apoptosis after incubation for 6 h with Rap, Baf, or their combination in H9c2/mock and H9c2/Pdcd1 cells under DOX-untreated basal condition. a Autophagy was evaluated using the autophagy LC3 HiBiT reporter assay. b Apoptosis was determined using a luciferase assay indicating increase in Annexin V-positive cells. Each value is expressed as a percentage relative to that in H9c2/mock control cells. Data are presented as the mean ± SEM of three samples. * p < 0.05, ** p < 0.01 vs. the control group in H9c2/mock cells; ## p < 0.01 vs. the control group in H9c2/Pdcd1 cells; † p < 0.05, †† p < 0.01 vs. the corresponding Rap-treated group

Article Snippet: Primary antibody against Pdcd1 was purchased from Proteintech (Rosemont, IL, USA).

Techniques: Incubation, Reporter Assay, Luciferase, Control

Journal: Cardiovascular Toxicology

Article Title: Overexpression of Programmed Cell Death 1 Prevents Doxorubicin-Induced Apoptosis Through Autophagy Induction in H9c2 Cardiomyocytes

doi: 10.1007/s12012-022-09726-w

Figure Lengend Snippet: Effects of Pdcd1 overexpression on doxorubicin (DOX)-induced apoptosis in human cancer cell lines, K562 (human erythroleukemia cells) and MCF-7 (human breast cancer cells). a Caspase-3/7 activity in the control (mock) and Pdcd1-overexpressing (Pdcd1) K562 cells was measured using a luciferase assay after incubation with 1 μM DOX for 8 h. The values were expressed as a percentage relative to that in DOX-untreated mock cells. b , c The apoptosis was assessed after a 24 h incubation with various concentrations of DOX (0.03–1 μM) using the Annexin V luciferase assay in K562 ( b ) and MCF-7 cells ( c ). Each value was expressed as a percentage relative to that in DOX-untreated control (mock) cells. d Quantification of apoptotic cells after a 24 h incubation with various concentrations of DOX (0.1–1 μM) in the control (mock) and Pdcd1-overexpressing MCF-7 cells. Nuclear morphological observations were performed after staining with H33342. e Control (mock) and Pdcd1-overexpressing (Pdcd1) MCF-7 cells stained with H33342 after a 24 h incubation with 1 μM DOX. Yellow arrows indicate a typical feature of apoptotic cells. Magnification = × 200. Scale bar = 50 μm. Data are presented as the mean ± SEM of three samples. * p < 0.05, ** p < 0.01 vs. the DOX-untreated mock (control) cells; ## p < 0.01 vs. the DOX-untreated Pdcd1-overexpressing cells; † p < 0.05, †† p < 0.01 vs. the corresponding DOX-treated mock cells

Article Snippet: Primary antibody against Pdcd1 was purchased from Proteintech (Rosemont, IL, USA).

Techniques: Over Expression, Activity Assay, Control, Luciferase, Incubation, Staining

Journal: Cardiovascular Toxicology

Article Title: Overexpression of Programmed Cell Death 1 Prevents Doxorubicin-Induced Apoptosis Through Autophagy Induction in H9c2 Cardiomyocytes

doi: 10.1007/s12012-022-09726-w

Figure Lengend Snippet: Schematic representation of the molecular mechanisms by which Pdcd1 overexpression plays a protective role against DOX-induced apoptosis and cell viability reduction in the cardiomyocyte H9c2 cells. Pdcd1-mediated signaling inhibits the expression of mTOR; and therefore, increases the expression of its downstream proteins, Atg3, Atg5, and Beclin-1, leading to the activation of autophagy, which inhibits the DOX-induced caspase activation and subsequent apoptosis. Therefore, Pdcd1 could be an effective molecule for cardioprotection from DOX-induced toxicity

Article Snippet: Primary antibody against Pdcd1 was purchased from Proteintech (Rosemont, IL, USA).

Techniques: Over Expression, Expressing, Activation Assay