Journal: Journal of Assisted Reproduction and Genetics

Article Title: Caspase signalling pathways in human spermatogenesis

doi: 10.1007/s10815-013-9938-8

Figure Lengend Snippet: Relative volumes of active caspases-8, −9 and −3 per germ cell stage per seminiferous tubules

Article Snippet: After 1 h of incubation with 5 % of normal horse serum (RT, Vectastain ABC Kit; diluted in 0.1 % Tween20/PBS), primary antibody was incubated overnight at 4 °C: active caspase-3 (Monoclonal Anti-Human/Mouse Cleaved Caspase-3 Antibody, R&D Systems, Minneapolis, USA; 1:150 diluted in 5 % normal horse serum); active caspase-9 (Monoclonal Anti-Caspase-9 Cleavage Site Specific, Sigma; 1:50 diluted in 5 % normal horse serum).

Techniques:

Journal: Redox Report : Communications in Free Radical Research

Article Title: Caffeine reduces oxidative stress to protect against hyperoxia-induced lung injury via the adenosine A2A receptor/cAMP/PKA/Src/ERK1/2/p38MAPK pathway

doi: 10.1080/13510002.2022.2143114

Figure Lengend Snippet: Caffeine regulates the cAMP/PKA/Src/ERK1/2/p38MAPK pathway. (A) qPCR was used to examine the mRNA levels of A2AR and PKA. (B) The levels of cAMP in each group. (C and D) Western blotting was used to examine the protein levels of A2AR, p-Src, p-ERK1/2, p-p38 MAPK and cleaved caspase-3. The data are presented as the mean ± SD ( n = 3). Compared to the 20%O 2 group, * P < 0.05. Compared to the 95%O 2 group, # P < 0.05. Compared to the caffeine and DMSO groups, & P < 0.05 and @ P < 0.05.

Article Snippet: After being blocked with 5% nonfat milk at 37°C for 1 h, the membranes were probed with anti-A2AR (Abcam, ab79714, USA), anti-p-Src (Abcam, ab185617, USA), anti-phospho-extracellular signal-regulated kinase-1/2 (anti-p-ERK1/2) (CST, #4370, USA), anti-phospho-P38 mitogen-activated protein kinases (anti-p-p38 MAPK) (Abcam, ab4822, USA), anti-cleaved caspase-3 (Bioss, bsm-33199M, China), or anti-β-actin (CST, #3700, USA) antibodies at 37°C for 1 h. Then, the membranes were washed three times and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature.

Techniques: Western Blot

Journal: Redox Report : Communications in Free Radical Research

Article Title: Caffeine reduces oxidative stress to protect against hyperoxia-induced lung injury via the adenosine A2A receptor/cAMP/PKA/Src/ERK1/2/p38MAPK pathway

doi: 10.1080/13510002.2022.2143114

Figure Lengend Snippet: A2AR regulates OS and cAMP/PKA/Src/ERK1/2/p38MAPK pathway-related proteins in a hyperoxia-induced AECs II model. AECs II were transfected with siA2AR and treated with hyperoxia for 24 h. (A and B) The levels of cAMP, GSH/GSSG ratio, ATP, SOD, GSH-PX and CAT. (C) qPCR was used to examine the mRNA levels of A2AR and PKA. (D and E) Western blotting was used to examine the protein levels of A2AR, p-Src, p-ERK1/2, p-p38 MAPK and cleaved caspase-3. The data are presented as the mean ± SD ( n = 3). Compared to the NC/20%O 2 -NC group, * P < 0.05. Compared to the 95%O 2 -NC group, # P < 0.05.

Article Snippet: After being blocked with 5% nonfat milk at 37°C for 1 h, the membranes were probed with anti-A2AR (Abcam, ab79714, USA), anti-p-Src (Abcam, ab185617, USA), anti-phospho-extracellular signal-regulated kinase-1/2 (anti-p-ERK1/2) (CST, #4370, USA), anti-phospho-P38 mitogen-activated protein kinases (anti-p-p38 MAPK) (Abcam, ab4822, USA), anti-cleaved caspase-3 (Bioss, bsm-33199M, China), or anti-β-actin (CST, #3700, USA) antibodies at 37°C for 1 h. Then, the membranes were washed three times and incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature.

Techniques: Transfection, Western Blot

Journal: Experimental Animals

Article Title: Acyl-CoA thioesterase 1 (ACOT1) overexpression alleviates heart failure by inhibiting oxidative stress and cardiomyocyte apoptosis through the Kelch-like ECH-associated protein1-NF-E2-related factor2 (KEAP1-NRF2) pathway

doi: 10.1538/expanim.24-0129

Figure Lengend Snippet: (A) The levels of MDA and GSH and the activity of SOD in myocardial tissues were analyzed by ELISA. (B) The protein levels and the quantification results of cleaved PARP, cleaved CASPASE-3 and cleaved CASPASE-9 in myocardial tissues. (C) ROS in myocardial tissues were detected by DHE staining (n=6). The fluorescence intensity was calculated to evaluate ROS levels. bar=50 µ m. # P <0.05, ## P <0.01, ### P <0.001. n=6. EP: empty plasmid; ACOT1 oe : ACOT1 overexpression.

Article Snippet: The following antibodies were used: cleaved CASPASE-3 (Affinity, Cincinnati, OH, USA, #AF7022) diluted in 1:1,000; cleaved CASPASE-9 (Affinity, #AF5240) diluted in 1:1,000; cleaved PARP (ABclonal, Wuhan, China, #A22535) diluted in 1:1,000; ACOT1 (Novus Biologicals, Littleton, CO, USA, #NBP3-10843) diluted in 1:500; KEAP1 (Affinity, #AF5266) diluted in 1:1,000; NRF2 (Affinity, #AF0639) diluted in 1:1,000; Histone H3 (Gene Tex, Alton Parkway Irvine, CA, USA, #GTX122148) diluted in 1:5,000; GAPDH (Proteintech, Wuhan, China, #60004-1-Ig) diluted in 1:10,000; Goat Anti-Rabbit IgG/HRP (Solarbio, #SE134) diluted in 1:3,000; Goat Anti-Mouse IgG/HRP (Solarbio, #SE131) diluted in 1:3,000.

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Staining, Fluorescence, Plasmid Preparation, Over Expression

Journal: Experimental Animals

Article Title: Acyl-CoA thioesterase 1 (ACOT1) overexpression alleviates heart failure by inhibiting oxidative stress and cardiomyocyte apoptosis through the Kelch-like ECH-associated protein1-NF-E2-related factor2 (KEAP1-NRF2) pathway

doi: 10.1538/expanim.24-0129

Figure Lengend Snippet: (A) Protein level and the quantification of ACOT1 in HL-1 cells with ACOT1 overexpression. (B) cell viability of HL-1 cells with hypoxia treatment. (C) The levels of MDA and GSH and the activity of SOD in cells. (D) The protein levels of cleaved PARP, cleaved CASPASE-3 and cleaved CASPASE-9 in cells and the quantification results. (E) TUNEL staining of HL-1 cells and the positive cell quantification, bar=100 µ m. (F) ROS levels in HL-1 cells were detected by flow cytometry, MFI: mean fluorescence intensity. ## P <0.01, ### P <0.001. n=3. EP: empty plasmid; ACOT1 oe : ACOT1 overexpression.

Article Snippet: The following antibodies were used: cleaved CASPASE-3 (Affinity, Cincinnati, OH, USA, #AF7022) diluted in 1:1,000; cleaved CASPASE-9 (Affinity, #AF5240) diluted in 1:1,000; cleaved PARP (ABclonal, Wuhan, China, #A22535) diluted in 1:1,000; ACOT1 (Novus Biologicals, Littleton, CO, USA, #NBP3-10843) diluted in 1:500; KEAP1 (Affinity, #AF5266) diluted in 1:1,000; NRF2 (Affinity, #AF0639) diluted in 1:1,000; Histone H3 (Gene Tex, Alton Parkway Irvine, CA, USA, #GTX122148) diluted in 1:5,000; GAPDH (Proteintech, Wuhan, China, #60004-1-Ig) diluted in 1:10,000; Goat Anti-Rabbit IgG/HRP (Solarbio, #SE134) diluted in 1:3,000; Goat Anti-Mouse IgG/HRP (Solarbio, #SE131) diluted in 1:3,000.

Techniques: Over Expression, Activity Assay, TUNEL Assay, Staining, Flow Cytometry, Fluorescence, Plasmid Preparation