Journal: Oncotarget

Article Title: Caspase-4 is essential for saikosaponin a-induced apoptosis acting upstream of caspase-2 and γ-H2AX in colon cancer cells

doi: 10.18632/oncotarget.22247

Figure Lengend Snippet: (A–C, E) SW480 cells were treated with 20 μM SSa for 15 h in the presence or absence of caspase-3 inhibitor (z-DEVD-fmk, labeled DEVD-f; 10 μM), caspase-8 inhibitor (z-IETD-fmk, labeled IETD-f; 10 μM), caspase-2 inhibitor (z-VDVAD-fmk, labeled VDVAD-f; 10 μM), or caspase-4 inhibitors (z-LEVD-fmk and Ac-LEVD-CHO, labeled LEVD-f and LEVD-C, respectively; each 10 μM). The activities of caspase-3 (A) , caspase-8 (B) , caspase-2 (C) , and caspase-4 (E) were monitored via the detection of pNA liberated from DEVD-pNA, IETD-pNA, VDVAD-pNA, and LEVD-pNA, respectively. Each column represents the mean ± standard error of three independent experiments. ** P < 0.01 compared with untreated controls. † P < 0.05 and †† P < 0.01 compared with SSa alone. ANOVA and Tukey’s test were used for statistical analysis. (D) SW480 cells were seeded and allowed to reach ∼30% confluence on the day of transfection. Cells were treated with siRNA to caspase-4 or scramble siRNA (each 20 nM) for 30 h, and then exposed to 20 μM SSa for 15 h. Caspase-4 knockdown efficiency was confirmed by Western blotting. Caspase-3, -8, and -2 activities were monitored as in Figure . Each column represents the mean ± standard error of three independent experiments. †† P < 0.01 compared with SSa alone. The Mann-Whitney U test was used for statistical analysis.

Article Snippet: Caspase-4 inhibitors, z-LEVD-fmk and Ac-LEVD-CHO, were obtained from Enzo Life Sciences (Plymouth Meeting, PA).

Techniques: Labeling, Transfection, Western Blot, MANN-WHITNEY

Journal: Oncotarget

Article Title: Caspase-4 is essential for saikosaponin a-induced apoptosis acting upstream of caspase-2 and γ-H2AX in colon cancer cells

doi: 10.18632/oncotarget.22247

Figure Lengend Snippet: (A and C) LoVo and SW480 cells were treated with 20 μM SSa for 30 h (for sub-G1 fraction analysis) or 24 h (for Hoechst 33342 staining) in the presence or absence of the caspase-4 inhibitors z-LEVD-fmk (LEVD-f; 10 μM) and Ac-LEVD-CHO (LEVD-C; 10 μM). (A) Cell cycle analysis was used to quantify apoptosis as the percentage of cells in the sub-G1 fraction. Each column represents the mean ± standard error from three experiments. ** P < 0.01 compared with untreated controls. † P < 0.05 and †† P < 0.01 compared with SSa alone. ANOVA and Tukey’s test were used for statistical analysis. (C) Morphologically apoptotic cells were quantified after Hoechst 33342 staining. Each column represents the mean ± standard error of cells with condensed/fragmented nuclei in three independent experiments. ** P < 0.01 compared with untreated controls. † P < 0.05 and †† P < 0.01 compared with SSa alone. ANOVA and Tukey’s test were used for statistical analysis. Representative images of Hoechst 33342 staining are shown. (B and D) LoVo and SW480 cells were seeded and allowed to reach ∼30% confluence on the day of transfection. Cells were treated with siRNA of caspase-4 (si-C4; 20 nM) or scrambled siRNA (si-Cont; 20 nM) for 30 h and then exposed to 20 μM SSa for 30 h (for sub-G1 fraction analysis) or 24 h (for Hoechst 33342 staining). Caspase-4 knockdown efficiency was confirmed via Western blotting. (B) Apoptosis was quantified as in Figure . Each column represents the mean ± standard error from three experiments. ** P < 0.01 compared with untreated controls. † P < 0.05 and †† P < 0.01 compared with SSa alone. ANOVA and Tukey’s test were used for statistical analysis. (D) Morphologically apoptotic cells were quantified as in Figure . Each column represents the mean ± standard error of three independent experiments. ** P < 0.01 compared with untreated controls. † P < 0.05 and †† P < 0.01 compared with SSa alone. ANOVA and Tukey’s test were used for statistical analysis. Representative images of Hoechst 33342 staining are shown. (E) LoVo and SW480 cells were treated with 15 μM SSa in the presence or absence of the caspase-4 inhibitors, z-LEVD-fmk (10 μM), and Ac-LEVD-CHO (10 μM). After 12 (for LoVo) or 14 (for SW480) days of incubation, cells were analyzed for cloning efficiency. The number of colonies was expressed as a percentage of the untreated control. Each column represents the mean ± standard error of three independent experiments. ** P < 0.01 compared with untreated controls. †† P < 0.01 compared with SSa alone. ANOVA and Tukey’s test were used for statistical analysis.

Article Snippet: Caspase-4 inhibitors, z-LEVD-fmk and Ac-LEVD-CHO, were obtained from Enzo Life Sciences (Plymouth Meeting, PA).

Techniques: Staining, Cell Cycle Assay, Transfection, Western Blot, Incubation, Clone Assay

Journal: Oncotarget

Article Title: Caspase-4 is essential for saikosaponin a-induced apoptosis acting upstream of caspase-2 and γ-H2AX in colon cancer cells

doi: 10.18632/oncotarget.22247

Figure Lengend Snippet: LoVo and SW480 cells were exposed to 20 μM SSa for 15 h in the presence or absence of z-LEVD-fmk (10 μM). (A) The percentage of cells positive for γ-H2AX foci was calculated as in Figure . Results are mean ± standard error for three experiments. ** P < 0.01 compared with untreated controls. † P < 0.05 and †† P < 0.01 compared with SSa alone. ANOVA and Tukey’s test were used for statistical analysis. (B) Western blotting was used to detect H2AX phosphorylation at Ser139. Blots are representative of three independent experiments, and data shown represent the mean ± standard error of three independent experiments. ** P < 0.01 compared with untreated controls. †† P < 0.01 compared with SSa alone. ANOVA and Tukey’s test were used for statistical analysis.

Article Snippet: Caspase-4 inhibitors, z-LEVD-fmk and Ac-LEVD-CHO, were obtained from Enzo Life Sciences (Plymouth Meeting, PA).

Techniques: Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Inflammation in Hypervolemic Hemodialysis Patients: The Roles of RelB and Caspase-4

doi: 10.3390/ijms242417550

Figure Lengend Snippet: Endotoxin levels and caspase-4 activity in normovolemic (N) and hypervolemic (H) patients: Endotoxin level in serum of N and H patients ( a ). Caspase-4 activity in N (N+LEVD) and H (H+LEVD) patients ( b ). Caspase-8 activity in N (N+IETD) and H (H+IETD) patients ( c ). Induction of caspase activity by indoxyl sulfate (IS) and specific inhibition of this caspase activity with caspase-4 specific inhibitor Ac-LEVD-CHO in N and H patients ( d ). Data were analyzed using Mann–Whitney tests or one-way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Cells were incubated for 20 h with or without the caspase-4 inhibitor (Ac-LEVD-CHO, 2 µM) and the caspase-8 inhibitor (Ac-IETD-CHO, 2 µM) (both from Merck Millipore, Darmstadt, Germany), respectively.

Techniques: Activity Assay, Inhibition, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Neutrophils drive endoplasmic reticulum stress-mediated apoptosis in cancer cells through arginase-1 release

doi: 10.1038/s41598-021-91947-0

Figure Lengend Snippet: Conditioned media from activated neutrophils induce apoptosis in HeLa cancer cells by activation of ER stress signals. ( a ) 1.5 × 10 7 neutrophils were stimulated with 100 nM fMLP, and the supernatants were incubated with HeLa cells for the indicated times, and then protein extracts were prepared and analyzed by Western blot. ( b ) Supernatant from fMLP-stimulated neutrophils (PMN-Spt) was incubated with HeLa cells for 48 h, which were previously untreated or pretreated for 1 h with 20 μM z-LEVD-fmk (caspase-4 inhibitor), and then analyzed for caspase-4 activation. ( c ) Supernatant from fMLP-stimulated neutrophils (PMN-Spt) was incubated with HeLa cells for 24 h, which were previously untreated or pretreated for 1 h with 100 μM z-IETD-fmk (caspase-8 inhibitor), and then analyzed for caspase-8 activation and Bap31 cleavage. ( d ) Induction of apoptosis determined by Annexin-V staining following incubation of HeLa cells with PMN-Spt for 24 h, in the absence or presence of z-VAD, z-LEVD or z-IETD (FlowJo X 10.0.7r2—proprietary commercial software— https://www.flowjo.com/ ). The data represented correspond to the mean ± SD of three independent experiments. Asterisks indicate significant differences. *** P < 0.001. ( e ) 1.5 × 10 7 neutrophils were stimulated with 100 nM of fMLP, and the supernatants (PMN-Spt) were incubated for 24 h with HeLa cells in the absence or presence of 50 μM nor-NOHA. Protein extracts were analyzed by Western blot for the indicated proteins. β-Actin was used as loading control. Molecular weights (in kilodaltons) of each protein are indicated at the right side of the panel. The gels were cropped to show the relevant sections. The results shown in ( a – c , e ) are representative of three independent experiments. (GraphPad Prism 8.0.1—proprietary commercial software— https://www.graphpad.com/ ).

Article Snippet: Caspase-4 inhibitor z-LEVD-fmk, and the broad pan-caspase inhibitor z-VAD-fmk were from Alexis Biochemicals (San Diego, CA).

Techniques: Activation Assay, Incubation, Western Blot, Staining, Software

Journal: Scientific Reports

Article Title: Neutrophils drive endoplasmic reticulum stress-mediated apoptosis in cancer cells through arginase-1 release

doi: 10.1038/s41598-021-91947-0

Figure Lengend Snippet: Conditioned media from activated neutrophils induce apoptosis in Bx-PC-3 pancreatic tumor cells by activation of ER stress signals. ( a , b ) 1.5 × 10 7 neutrophils were stimulated with 100 nM fMLP, and the supernatant was incubated with the BxPC-3 cell line for the indicated times. Then, protein extracts were prepared and analyzed by Western blot. ( c , d ) Supernatants from fMLP-stimulated neutrophils (PMN-Spt) were incubated with BxPC-3 pancreatic cancer cells for 24 h, which were previously untreated or pretreated for 1 h with 20 μM z-LEVD-fmk (caspase-4 inhibitor) or 100 μM z-IETD-fmk (caspase-8 inhibitor), and then analyzed for caspase-4 activation ( c ), or caspase-8 activation and Bap31 cleavage ( d ). β-Actin was used as loading control. Molecular weights (in kilodaltons) of each protein are indicated at the right side of each panel. The results shown in ( a – d ) are representative of three independent experiments. The gels were cropped to show the relevant sections. ( e ) Induction of apoptosis determined by Annexin-V staining following incubation of BxPC-3 cells with PMN-Spt for 24 h, in the absence or presence of z-VAD, z-LEVD or z-IETD (FlowJo X 10.0.7r2—proprietary commercial software— https://www.flowjo.com/ ). The data correspond to the mean ± SD of at least three independent experiments. Asterisks indicate significant differences. *** P < 0.001. (GraphPad Prism 8.0.1—proprietary commercial software— https://www.graphpad.com/ ).

Article Snippet: Caspase-4 inhibitor z-LEVD-fmk, and the broad pan-caspase inhibitor z-VAD-fmk were from Alexis Biochemicals (San Diego, CA).

Techniques: Activation Assay, Incubation, Western Blot, Staining, Software

Journal: Scientific Reports

Article Title: Neutrophils drive endoplasmic reticulum stress-mediated apoptosis in cancer cells through arginase-1 release

doi: 10.1038/s41598-021-91947-0

Figure Lengend Snippet: Schematic model of ER involvement in the induction of apoptosis in cancer cells following release of arginase-1 from human neutrophils. This is a schematic diagram to portray a plausible mechanism of how neutrophils can induce apoptosis in tumor cells following arginase-1 (ARG1) release. ARG1 is released from human neutrophils, either after cell activation leading to exocytosis of granule contents or after cell death. ARG1 leads to L-Arg depletion in the surrounding medium, and this induces an ER stress response and apoptosis in cancer cells. During this process, caspase-4 and -8 are activated, and there is an ER-mitochondria interplay mediated by the action of caspase-8 on Bap31. See text for details. Source: Own elaboration.

Article Snippet: Caspase-4 inhibitor z-LEVD-fmk, and the broad pan-caspase inhibitor z-VAD-fmk were from Alexis Biochemicals (San Diego, CA).

Techniques: Activation Assay