Journal: Frontiers in Pharmacology

Article Title: Anti-Myocardial Ischemia Reperfusion Injury Mechanism of Dried Ginger-Aconite Decoction Based on Network Pharmacology

doi: 10.3389/fphar.2021.609702

Figure Lengend Snippet: Experimental validation of key signaling pathways and mitochondrial key targets in vivo . Dried ginger-aconite decoction(DAD) activates the PI3k/Akt/GSK-3β signaling pathway and inhibits the expression of Cyt-C and CASP9. Data were presented as mean standard deviation (SD) of three independent experiments. # p < 0.05, ## p < 0.01 vs. sham group. * p < 0.05, ** p < 0.01 vs. MI/RI group.

Article Snippet: Cyt-C, β -action, GADPH, Casp9, PI3K, AKT, Bax, Bcl-2 and phosphorylated( P )-AKT, GSK3β were also purchased from Boster Biological Technology Co., Ltd. (Wuhan, China).

Techniques: Biomarker Discovery, Protein-Protein interactions, In Vivo, Expressing, Standard Deviation

Journal: Cancer Cell International

Article Title: Silencing Uracil-DNA glycosylase inhibits colorectal cancer progression

doi: 10.1186/s12935-025-04089-y

Figure Lengend Snippet: UNG activated several signaling pathways in CRC cells. Western blot was performed to detect the levels of mTOR, p-mTOR, p70 S6K, p-P70 S6K, AKT, p-AKT, AMPK, p-AMPK, ERK, p-ERK, Bax, Bcl2, cleavages of caspase-9, and caspase-3 in UNG-knockdown CRC cells. Fold changes (Fc) are shown below the bars

Article Snippet: The membranes were incubated overnight at 4 °C with the following primary antibodies: UNG, P70 S6K, p-P70 S6K(S424) (1:1000, Bioworld), β-actin (1:1000, Abcam), cleaved caspase-3, AMPKα1/AMPKα2, p-AMPKα1(Thr183)/AMPKα2(Thr172) (1:1000, Beyotime), BAX, BCL2, AKT1/2/3, mTOR, and p-mTOR (Ser2448) (1:1000; BOSTER), cleaved caspase-9, p-AKT(Ser473), ERK1/2, and p-ERK1/2(Thr202/Tyr204)/(Thr185/Tyr187) (1:1000; ZEN BIO), and GAPDH (1:1000; Proteintech).

Techniques: Protein-Protein interactions, Western Blot, Knockdown

Journal: British Journal of Cancer

Article Title: Mechanism of action for N-substituted benzamide-induced apoptosis

doi: 10.1038/sj.bjc.6600136

Figure Lengend Snippet: Induction of apoptosis in 70Z/3 cells treated with 3CPA at 250 μ M and 500 μ M . ( A ) Cytochrome c release into the cytosol after exposure to 3CPA for indicated time periods measured by Western blot analysis. ( B ) Pro-caspase-9 processing determined in cytosolic fractions after exposure to 3CPA. Actin was used as an internal standard of the protein load and the results shown are representatives of two separate experiments. ( C ) Caspase inhibitors reduce 3CPA-induced apoptosis in 70Z/3 cells. Apoptosis, measured as AnnexinV + 7AAD − cells, was determined after exposure to 3CPA at 250 μ M (left) and 500 μ M (right) for 24 h. The pan-caspase inhibitor ZVAD-fmk (50 μ M , n =4), the caspase-9 inhibitor LEHD-fmk (50 μ M , n =3), or the caspase-8 inhibitor IETD-fmk (50 μ M , n =3) were added 1 h before treatment with 3CPA. The level of apoptosis is expressed relative to the level in cell cultures treated with the inhibitor only. Results show mean±s.d., and (*) indicates a statistical difference compared to 3CPA only (paired t -test, P <0.05).

Article Snippet: To detect pro-caspase-9 or active caspase-9, a rabbit polyclonal anti-mouse caspase-9 (Santa Cruz) or a rabbit polyclonal anti-human caspase-9 (Pharmingen) was used.

Techniques: Western Blot

Journal: British Journal of Cancer

Article Title: Mechanism of action for N-substituted benzamide-induced apoptosis

doi: 10.1038/sj.bjc.6600136

Figure Lengend Snippet: Over-expression of Bcl-2 in 70Z/3 cells inhibits 3CPA-induced apoptosis. ( A ) FACS-analysis of 70Z/3 (upper panel) and 70Z/3 Bcl-2+ (lower panel) cells after exposure to 3CPA (500 μ M ) for 18 h. 7AAD negative (viable) cells were gated (gate A) and analysed for Annexin V + expression (% of gated cells). ( B ) Levels of Bcl-2 and cytochrome c expression were determined by Western blot analysis in mitochondrial enriched fractions after 18 h incubation with 3CPA at the indicated concentrations (30 μg protein per lane). ( C ) Pro-caspase-9 processing into active caspase-9 was determined in the cytosolic fraction after 18 h incubation with 3CPA at the indicated concentrations. Actin was used as an internal standard to control the amount of cytosolic protein loaded in each lane. Results shown are representatives of two independent experiments.

Article Snippet: To detect pro-caspase-9 or active caspase-9, a rabbit polyclonal anti-mouse caspase-9 (Santa Cruz) or a rabbit polyclonal anti-human caspase-9 (Pharmingen) was used.

Techniques: Over Expression, Expressing, Western Blot, Incubation

Journal: British Journal of Cancer

Article Title: Mechanism of action for N-substituted benzamide-induced apoptosis

doi: 10.1038/sj.bjc.6600136

Figure Lengend Snippet: N-substituted benzamides induce cytochrome c release and activation of caspase-9 in HL60 cells. ( A ) Cytochrome c release induced by MCA (500 μ M ) and 3CPA (250 μ M ) in HL60 cells after 24 and 48 h, measured by Western blot analysis of mitochondria or cytosol fractions (30 μg protein/lane). Cells were incubated with etoposide for 6 and 18 h at 100 μ M as a positive control. ( B ) Activation of caspase-9 after treatment of HL60 cells with 3CPA at 250 and 500 μ M for 12 and 18 h (48 μg protein per lane). One representative experiment out of two is shown.

Article Snippet: To detect pro-caspase-9 or active caspase-9, a rabbit polyclonal anti-mouse caspase-9 (Santa Cruz) or a rabbit polyclonal anti-human caspase-9 (Pharmingen) was used.

Techniques: Activation Assay, Western Blot, Incubation, Positive Control

Journal:

Article Title: Structure-based discovery of an organic compound that binds Bcl-2 protein and induces apoptosis of tumor cells

doi:

Figure Lengend Snippet: Apoptosis induced by HA14-1 in HL-60 cells as indicated by DNA ladders (A) and cleavage of caspase-9, caspase-3 and PARP (B). DNA ladders were visualized on 2% of agarose gel after the cells were treated with 50 μM HA14-1 compound at the designated time. M, DNA marker. Lanes: 1, control; 2, 1 h of HA14-1 treatment; 3, 2 h of HA14-1 treatment; 4, 3 h of HA14-1 treatment; 5, 4 h of HA14-1 treatment; 6, preincubation with zVAD-fmk for 2 h before the treatment with HA14-1 for 4 h. The cleavage of caspase-3, -9, and PARP was shown by Western blot analysis using polyclonal rabbit antibodies.

Article Snippet: Antibodies to detect the cleavage of caspase-3 (PharMingen, catalog no. 65906E; diluted to 1:1,000), caspase-9 (PharMingen, catalog no. 68086E; diluted to 1:1,000), and poly(ADP-ribose)-polymerase (PARP) (Boehringer Mannheim, catalog no. 1835238; diluted to 1:2,000) were used for 1 h incubation at room temperature.

Techniques: Agarose Gel Electrophoresis, Marker, Western Blot

Journal: Investigational New Drugs

Article Title: The molecular mechanism of anticancer action of novel octahydropyrazino[2,1-a:5,4-a′]diisoquinoline derivatives in human gastric cancer cells

doi: 10.1007/s10637-018-0584-y

Figure Lengend Snippet: Immunofluorescence analysis of caspase 9 expression and translocation in human gastric AGS cells treated with different concentrations of the tested compounds ( 1, 2 ) and etoposide

Article Snippet: The cells were rinsed and incubated with anti-caspase-9 mouse monoclonal antibody (BD Biosciences, San Jose, CA, USA; 1:200), or anti-caspase-3 (Ab-2) mouse monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA; 1:200), or anti- p53 (DO-1) mouse monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA, 1:200), or anti-p-AKT (anti-phospho-PKB (pThr 308 ) rabbit polyclonal antibody (Sigma-Aldrich, St. Louis, MO, USA; 1:200) or anti-ERK1/ERK2 (pT202/pY204) mouse monoclonal antibodies (BD Biosciences, San Jose, CA, USA; 1:200) for 1 h at room temperature.

Techniques: Immunofluorescence, Expressing, Translocation Assay