active caspase8  (Cell Signaling Technology Inc)

Journal: Journal of Ginseng Research

Article Title: 20( S )-ginsenoside Rh2 induces caspase-dependent promyelocytic leukemia-retinoic acid receptor A degradation in NB4 cells via Akt/Bax/caspase9 and TNF-α/caspase8 signaling cascades

doi: 10.1016/j.jgr.2020.05.001

Figure Lengend Snippet: GRh2 induced mitochondrial damage and caspase activation in NB4 cells. NB4 cells were incubated with 30 μM, 40 μM, and 50 μM GRh2 for 12 h. (A) JC-1 staining was used to observe changes in mitochondrial membrane potential of NB4 cells after administering GRh2 Scale bar=100 μm. Red fluorescence indicates a higher membrane potential, and green fluorescence represents a membrane potential dissipation. (B) The intracellular ROS level of NB4 cells after administering GRh2 was detected via fluorescent-probe DCFH-DA, and a representative picture of three replicates is shown Scale bar=500 μm. (C) Quantitative statistical graph of relative fluorescence intensity of intracellular DCF. The result are shown as the mean ± SD (n = 3) # p < 0.05, ∗∗∗ p < 0.001 versus solvent. (D) Protein expression levels of pro-caspase3, cleaved-caspase3, Bax, Bcl-2, and cleaved-PARP in NB4 cells were detected via Western Blot. A representative picture of three replicates is shown. (E) Quantitative statistical graph of the relative expression levels of proteins. The results are shown as the mean ± SD (n = 3) ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (F) Quantitative statistical graph of caspase8 and caspase9 cleavage activation levels in NB4 cells after administering of GRh2, Ac-IETD-pNA, and Ac-LEHD-pNA methods were used. The results are shown as the mean ± SD (n = 3) ∗∗∗ p < 0.001 versus solvent (caspase8), ### p < 0.001 versus solvent (caspase9). GRh2, 20( S)-ginsenoside Rh2; ROS, reactive oxygen species.

Article Snippet: Caspase8 and caspase9 enzyme activity assay kits (Beyotime Biotechnology Co. Ltd.) were used to detect caspase8 and caspase9 enzyme activity.

Techniques: Activation Assay, Incubation, Staining, Membrane, Fluorescence, Solvent, Expressing, Western Blot

Journal: Journal of Ginseng Research

Article Title: 20( S )-ginsenoside Rh2 induces caspase-dependent promyelocytic leukemia-retinoic acid receptor A degradation in NB4 cells via Akt/Bax/caspase9 and TNF-α/caspase8 signaling cascades

doi: 10.1016/j.jgr.2020.05.001

Figure Lengend Snippet: GRh2 activated the TNF-α/caspase8 cascade. (A) NB4 cells were incubated with 30 μM, 40 μM, or 50 μM GRh2 for 12 h. Protein expression levels of FasL, Fas, TNF-α, and TNFR1 in NB4 cells were detected via Western blot. A representative picture of three replicates is shown. (B) Quantitative statistical graph of the relative protein expression levels. The results are shown as the mean ± SD (n = 3) ∗ p < 0.05, ∗∗ p < 0.01. (C) RT-PCR was used to detect the mRNA expression level of TNF-α in NB4 cells after GRh2 administration. The results are shown as the mean ± SD (n = 3) ∗∗ p < 0.01, ∗∗∗ p < 0.001 versus solvent. After preincubation with 1.5 μM TNF-α inhibitor, C 87, for 2 h, 30 μM, 40 μM, or 50 μM GRh2 was applied for another 12 h. (D) CCK-8 assay measured the NB4 cell viability. The results are shown as the mean ± SD (n = 6) ∗∗∗ p < 0.001, ### p < 0.001. (E) Hoechst 33258 staining was used to observe changes in nuclear morphology of NB4 cells after GRh2 and C 87 administration Scale bar=200 μm. (F) Quantitative statistical graph of caspase8 cleavage activation levels in NB4 cells. The Ac-IETD-pNA method was used. The results are shown as the mean ± SD (n = 3) ∗∗∗ p < 0.001, # p < 0.05. GRh2, 20( S)-ginsenoside Rh2.

Article Snippet: Caspase8 and caspase9 enzyme activity assay kits (Beyotime Biotechnology Co. Ltd.) were used to detect caspase8 and caspase9 enzyme activity.

Techniques: Incubation, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Solvent, CCK-8 Assay, Staining, Activation Assay