Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Hypoxia-Inducible Factor 1-α (HIF-1α) Induces Apoptosis of Human Uterosacral Ligament Fibroblasts Through the Death Receptor and Mitochondrial Pathways

doi: 10.12659/MSM.913384

Figure Lengend Snippet: Expression of death receptor-associated apoptotic markers in the cobalt chloride (CoCl 2 )-treated human uterosacral ligament fibroblasts (hUSLFs). ( A ) Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), Fas, decoy receptor 2 (DcR2), cellular FLICE inhibitory protein (c-FLIP), death receptor 5 (DR5), and cleaved caspase-8 protein levels by Western blot analysis in hUSLFs treated with the chemical inducer of hypoxia, cobalt chloride (CoCl 2 ), at 48 h. ( B ) Quantitative analysis of Fas, TRAIL, DcR2, c-FLIP, and DR5 expression standardized to β-actin. ( C ) Caspase-8 activity measured in hUSLFs treated with CoCl 2 for 48 h. ( D ) After 48 h, the effects of CoCl 2 on the levels of lactate dehydrogenase (LDH) in hUSLFs were measured. ( E ) Results of the MTT assay for cell viability following treatment with CoCl 2 for 48 h with or without the presence of 50 μM of the caspase-8 inhibitor, Z-IETD-FMK. * P <0.05, ** P <0.01. Con – control. C-cas-8 – cleaved caspase-8.

Article Snippet: After pretreated with and without 50 μM of the caspase-8 inhibitor, Z-IETD-FMK (MedChem Express, Monmouth Junction, NJ, USA), and treatment with and without 100 μM of the caspase-9 inhibitor, Z-LEHD-FMK (KeyGen Biotech Co. Ltd., Nanjing, China) for 2 h, cells were treated with CoCl 2 48 h later.

Techniques: Expressing, Western Blot, Activity Assay, MTT Assay, Control

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Hypoxia-Inducible Factor 1-α (HIF-1α) Induces Apoptosis of Human Uterosacral Ligament Fibroblasts Through the Death Receptor and Mitochondrial Pathways

doi: 10.12659/MSM.913384

Figure Lengend Snippet: Expression of mitochondrial-associated apoptotic markers in the cobalt chloride (CoCl 2 )-treated human uterosacral ligament fibroblasts (hUSLFs). ( A ) Bcl-2, Bax, cytochrome C, Bcl-2 interacting protein 3 (BNIP3), cleaved caspase-3, and cleaved caspase-9 protein levels were analyzed by Western blot in human uterosacral ligament fibroblasts (hUSLFs) that were treated with the chemical inducer of hypoxia, cobalt chloride (CoCl 2 ) at 48 h. ( B ) Quantitative analysis of Bcl-2/Bax, cytochrome C, BNIP3, cleaved caspase-3, and cleaved caspase-9 protein expression standardized to β-actin. ( C ) Caspase-3, and caspase-9 activity measured in hUSLFs that were treated with CoCl 2 for 48 h. ( D ) Results of the MTT assay for cell viability following treatment with CoCl 2 for 48 h, which were also exposed to 100 μM of the caspase-9 inhibitor, Z-LEHD-FMK, with or without the presence of 50 μM of the caspase-8 inhibitor, Z-IETD-FMK. ( E ) Photomicrograph of JC-1 fluorescence mitochondrial imaging used to study the change in mitochondrial membrane potential in the different groups. ( F ) The relative mitochondrial membrane potential level was evaluated by comparing the red fluorescence cells to green fluorescence cells in the groups. * P <0.05, ** P <0.01. Scale bar=100 μm. Con – control; C-cas-3 – cleaved caspase-3; C-cas-9 – cleaved caspase-9; Cyto c – cytochrome C.

Article Snippet: After pretreated with and without 50 μM of the caspase-8 inhibitor, Z-IETD-FMK (MedChem Express, Monmouth Junction, NJ, USA), and treatment with and without 100 μM of the caspase-9 inhibitor, Z-LEHD-FMK (KeyGen Biotech Co. Ltd., Nanjing, China) for 2 h, cells were treated with CoCl 2 48 h later.

Techniques: Expressing, Western Blot, Activity Assay, MTT Assay, Fluorescence, Imaging, Membrane, Control

Journal: Cell Death & Disease

Article Title: Downregulation of Engulfment and cell motility 1 (Elmo1) induces quiescence and resistance to poly(I:C)-induced apoptosis in endothelial cells

doi: 10.1038/s41419-025-08341-1

Figure Lengend Snippet: a Transcript levels of selected genes associated with apoptosis before (0 h) and after (24 h) the poly(I:C) treatment. Transcript levels of each gene are shown as TPM (transcript per million). Data are mean ± SE ( N = 3). * p < 0.05, ** p < 0.01, *** p < 0.001, n.s . not significant. b Representative images of Mock, siNT and siELMO1 cells 24 h after the poly(I:C) stimulation. Cells were treated with or without 20 ng/ml of caspase-8 inhibitor Z-IETD-FMK throughout the experiment. Bar = 20 μm. c Cell death in (b) was quantified as in Fig. . Data are mean ± SD ( N = 3). *** p < 0.001. d Western blot of full-length and cleaved caspase-8, -3 and -7 before (0 h) and after (4 h, 8 h) the stimulation with poly(I:C). Beta-Actin was detected as a loading control. Images are representative of three independent experiments.

Article Snippet: The absence of mycoplasma contamination was confirmed using the Mycoplasma PCR Detection Kit (Abcam, Cambridge, MA, USA), Caspase-8 inhibitor Z‐IETD‐FMK was purchased from R&D systems (Minneapolis, MN, USA).

Techniques: Western Blot, Control

Journal:

Article Title: Daxx Silencing Sensitizes Cells to Multiple Apoptotic Pathways

doi: 10.1128/MCB.23.20.7108-7121.2003

Figure Lengend Snippet: Involvement of caspases in Daxx silencing-mediated apoptosis. (A) TUNEL assay showing synergistic induction of apoptosis by siDaxx and UV. HeLa cells were transfected with siDaxx or control reagent for 48 h. The caspase inhibitor z-VAD-fmk (z-VAD, 20 μM) was added 1 h prior to UV irradiation (50 J/m2). Apoptosis was determined by TUNEL (red) and DAPI (blue) staining 8 h after UV irradiation. (B) Effect of various caspase inhibitors on siDaxx-mediated apoptosis. HeLa cells were transfected with siC (lanes 1) or siDaxx (lanes 2 to 6), followed by treatment with actinomycin D (AD, 100 nM), UV irradiation (50 J/m2), or TNF-α (10 ng/ml) in the absence (lanes 2) or presence (lanes 3 to 6) of various caspase inhibitors. Cells were treated with the general caspase inhibitor z-VAD-fmk (lanes 3), the caspase 1 inhibitor z-YVAD-fmk (lanes 4), the caspase 8 inhibitor z-IETD-fmk (lanes 5), or the caspase 9 inhibitor z-LEHD-fmk (lanes 6). Apoptotic cells were detected by TUNEL assay and counting at least 200 cells in three different fields. The general caspase inhibitor z-VAD-fmk completely blocked siDaxx-enhanced apoptosis, while z-IETD-fmk and z-LEHD-fmk partially suppressed siDaxx-enhanced apoptosis.

Article Snippet: The general caspase inhibitor z-VAD-fmk, the caspase 1 inhibitor z-YVAD-fmk, the caspase 8 inhibitor z-IETD-fmk, and the caspase 9 inhibitor z-LEHD-fmk were purchased from BD Biosciences Clontech (Palo Alto, Calif.).

Techniques: TUNEL Assay, Transfection, Irradiation, Staining

Journal:

Article Title: Daxx Silencing Sensitizes Cells to Multiple Apoptotic Pathways

doi: 10.1128/MCB.23.20.7108-7121.2003

Figure Lengend Snippet: Synergistic activation of caspases by Daxx silencing. (A) Effects of siDaxx on cleavage of caspase 8, caspase 9, and PARP. HeLa cells with (+) or without (−) siDaxx transfection were treated with anti-Fas antibody (200 ng/ml), UV (50 J/m2), or actinomycin D (AD, 100 nM). Proteolytic cleavage of caspase 8 (Casp-8), caspase 9 (Casp-9), and PARP was determined by Western blotting 6 h after the treatment. Arrows at the right mark the proteolytic fragments. Protein molecular size markers are shown at left (in kilodaltons). Daxx silencing synergistically activated caspase 8, caspase 9, and PARP cleavage with anti-Fas antibody, UV, and actinomycin D. These proteolytic cleavages were inhibited by the general caspase inhibitor z-VAD-fmk (z-VAD, 20 μM), but not by the caspase 1 inhibitor z-YVAD-fmk (z-YVAD, 20 μM). (B) Kinetics of caspase activation in siDaxx-treated cells. Control siC or siDaxx-transfected HeLa cells were treated with anti-Fas antibody (200 ng/ml), TNF-α (10 ng/ml), TNF-α (2 ng/ml) plus cycloheximide (Chx, 2 μg/ml), actinomycin D (100 nM), or UV (50 J/m2), and cells were harvested at the time points shown at the bottom. The harvested cells were analyzed by Western blotting for proteolytic processing of caspase 8 and caspase 9 and by TUNEL assay for percent apoptosis. Boxes at the bottom mark the apparent time points of caspase activation and apoptosis.

Article Snippet: The general caspase inhibitor z-VAD-fmk, the caspase 1 inhibitor z-YVAD-fmk, the caspase 8 inhibitor z-IETD-fmk, and the caspase 9 inhibitor z-LEHD-fmk were purchased from BD Biosciences Clontech (Palo Alto, Calif.).

Techniques: Activation Assay, Transfection, Western Blot, TUNEL Assay

Journal:

Article Title: Daxx Silencing Sensitizes Cells to Multiple Apoptotic Pathways

doi: 10.1128/MCB.23.20.7108-7121.2003

Figure Lengend Snippet: Potential mechanisms of the antiapoptotic function of Daxx. Daxx might inhibit Fas-mediated caspase 8 activation and UV- or stress-induced JNK activation. This ability might be due to association of Daxx with the cytoplasmic domain of Fas. Consequently, depletion of Daxx stimulated caspase 8 and JNK activation, causing early mitochondrial cytochrome c release and activation of caspase 9 and caspase 3. In addition, PML may sequester Daxx to PODs, inhibiting its transcriptional repressor function. The oncogenic fusion protein PML-retinoic acid receptor alpha (RARα) may disrupt PODs, causing the release of Daxx from PODs and its association with condensed chromatin. We hypothesize that the association of Daxx with condensed chromatin might cause repression of selected caspase genes. Consequently, depletion of Daxx may increase the expression of apoptotic proteins, resulting in sensitization of cells to induction of apoptosis.

Article Snippet: The general caspase inhibitor z-VAD-fmk, the caspase 1 inhibitor z-YVAD-fmk, the caspase 8 inhibitor z-IETD-fmk, and the caspase 9 inhibitor z-LEHD-fmk were purchased from BD Biosciences Clontech (Palo Alto, Calif.).

Techniques: Activation Assay, Expressing