Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Caspase-3 cleaves and activates the NADase SARM1 to promote apoptosis, linking two cell death mechanisms

doi: 10.1073/pnas.2528118123

Figure Lengend Snippet: SARM1 promotes apoptotic cell death. ( A ) Neuro-2a cells show defective apoptosis. WT Neuro-2a cells and two single-cell derived Sarm1 KO lines (KO-1 and KO-2, generated by CRISPR-Cas9 mediated targeting) were treated either with DMSO or with TNF-α (20 ng/mL, TNF) combined with cycloheximide (10 µg/mL, CHX) for 16 h. Percent cell survival was determined by measuring total ATP level and normalizing to control (DMSO) treatment. Data are from three biological replicates. The two KO lines were verified by Western blotting of SARM1 ( Left ) using a monoclonal antibody targeting the amino terminus of SARM1 (clone 10G2, SI Appendix , Fig. S4 ). Tubulin Western blot was used as loading control. Western blot data shown are representative of three independent experiments. ( B ) Caspase-3/7 activity (i.e., DEVDase activity) following TNF and CHX cotreatment was measured using the caspase-glo kit from Promega. AU, arbitrary units. Data are from three biological replicates. ( C and D ) effect of SARM1 on apoptosis of macrophages and T cells. ( C ) primary bone marrow derived macrophages were treated with DMSO or TNF (10 ng/mL) and CHX (10 µg/mL) for 7 h. ( D ) primary T cells were treated with ABT-737 (1 µM) for 23 h. Percent cell survival was determined by measuring total ATP level and normalized to control (DMSO) treatment. Data are from three biological replicates. ( E ) SARM1 expression sensitizes HeLa cells to apoptosis. HeLa cells stably expressing GFP (HeLa+GFP) and HeLa cells stably expressing SARM1 (HeLa+SARM1) were treated with TNF+CHX for 18 h. Percent cell survival was determined by measuring total ATP level and normalized to control (DMSO) treatment. Data are from three biological replicates. SARM1 expression was confirmed by Western blot ( Left ). A portion of the stain free gel image was used as an internal loading control. Western blot data shown are representative of three independent experiments. ( F and G ) Sarm1 KO blocks anti-NGF-induced axon degeneration in the presence of caspase inhibitor. ( F ) Representative images of WT and Sarm1 KO DRG cultures (7 d in vitro, DIV) treated with DMSO, anti-NGF, or anti-NGF together with zVAD-FMK (100 µM, zVAD) for 22 h. (Scale bar, 50 µm.) Images are representative of three independent experiments. ( G ) Axon degeneration percentage of ( F ) was quantified and shown in the bar graphs. Data are from three biological replicates. In all panels, Student’s t test was used to determine statistical significance. * Indicates P < 0.05, NS, nonsignificant. Error bars represent SEM.

Article Snippet: ABT-737 and the caspase inhibitor zVAD-FMK were purchased from Selleck Chemicals.

Techniques: Derivative Assay, Generated, CRISPR, Control, Western Blot, Activity Assay, Expressing, Stable Transfection, Staining, In Vitro

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Caspase-3 cleaves and activates the NADase SARM1 to promote apoptosis, linking two cell death mechanisms

doi: 10.1073/pnas.2528118123

Figure Lengend Snippet: SARM1 is cleaved by active caspase-3. ( A ) DRG cultures were treated with anti-NGF for 16 h. Western blot of SARM1 protein shows reduction of full-length SARM1 (SARM1 FL) and increase of a band of ~33 kDa. A Sarm1 KO sample was used to demonstrate the specificity of the SARM1 antibody ( Left lane). A portion of the stain free gel image was used as an internal loading control. Data are representative of three independent experiments. ( B ) SARM1 from DRG cultures treated with ABT-737 (10 µM, 6 h) also shows a decrease in full-length SARM1 and increase in the ~33 kDa band. A portion of the stain-free gel image was used as an internal loading control. Data are representative of three independent experiments. ( C ) Western blot of SARM1 from control DRG cultures and following axotomy (6 h) shows that SARM1 is not cleaved during Wallerian degeneration. A portion of the stain free gel image was used as an internal loading control. Data are representative of three independent experiments. ( D ) Neuro-2a cells were treated with DMSO (control) or with TNF+CHX for 18 h to induce apoptosis. As in primary neurons, there is a reduction of full-length SARM1 and increase of a band of ~33 kDa. Both effects are blocked by the pan-caspase inhibitor zVAD. T + C: TNF+CHX, T + C + Z: TNF+CHX+zVAD. (Full Western blot shown in SI Appendix , Fig. S2 ). Tubulin Western blot was used as loading control. Data are representative of three independent experiments. ( E ) In vitro cleavage of recombinant SARM1 by active caspase-3. Purified SARM1 (on beads) was incubated with active caspase-3. Cleavage was determined by protein electrophoresis and Coomassie staining. *HC, anti-Flag antibody heavy chain conjugated on beads, *LC: anti-Flag antibody light chain conjugated on beads. Data are representative of three independent experiments. ( F ) Western blot of the caspase-cleaved SARM1 samples using antibody targeting the N-terminal FLAG tag. Data are representative of three independent experiments. Diagram shows the location of the FLAG tag at the N terminus of SARM1. ( G ) Effect of the SARM1 D314A mutation on caspase-3 cleavage. Recombinant WT SARM1 or SARM1 D314A were incubated with active caspase-3 and cleavage was determined as in E . Data are representative of three independent experiments. ( H ) Effect of SARM1 catalytic mutant on caspase-3 cleavage. Data are representative of three independent experiments.

Article Snippet: ABT-737 and the caspase inhibitor zVAD-FMK were purchased from Selleck Chemicals.

Techniques: Western Blot, Staining, Control, In Vitro, Recombinant, Purification, Incubation, Protein Electrophoresis, FLAG-tag, Mutagenesis

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Caspase-3 cleaves and activates the NADase SARM1 to promote apoptosis, linking two cell death mechanisms

doi: 10.1073/pnas.2528118123

Figure Lengend Snippet: Cleavage activates SARM1. ( A ) Cell death–inducing activity of SARM1 truncations and caspase-3 cleaved fragments. SARM1 fragments, diagrammed on the left, were transiently transfected into HEK293T cells. Cell survival was measured 48 h later using the CellTiter-Glo Luminescent Cell Viability Assay. Percent cell survival was determined by normalization to control (EGFP expressing vector) transfection. Data are from three biological replicates. MLS, mitochondrial localization signal. ARM, armadillo repeats. SAM, sterile alpha motif. TIR, Toll, Interleukin 1 receptor, and Resistance protein domain. ( B ) The effect of SARM1 cleavage on the SARM1 NADase activity. Recombinant SARM1 (lacking its N-terminal 27 amino acids, i.e., SARM1 delta27) was incubated with active caspase-3 (1 unit) or control for 2 h at 37 °C. NADase activity was determined using the ε-NAD assay. AU, arbitrary units. Data are from three biological replicates. The cleavage of SARM1 delta27 was verified using a stain free gel. Stain free gel image was representative of three biological replicates. ( C ) Electroporation of PP triggers cell death of cells expressing engineered SARM1(with PP site, engineered EVLFNGP after D314) but not WT SARM1. EGFP, WT SARM1 or SARM1 PP expressing plasmids were transfected into HEK293T cells for 24 h, then control protein BSA or PP was electroporated into the cells. Percent cell survival was determined 20 h after electroporation by normalizing total ATP level to control electroporation. Data are from three biological replicates. ( D ) Electroporation of PP triggers the cleavage in HEK293T cells of engineered SARM1 containing a PP site (SARM1 PP) but not WT SARM1. SARM1 Western blot image was representative of three biological replicates. ( E ) Diagram of the findings. The caspase-3 cleavage site was determined by Edman degradation of the C-terminal fragment. In all panels, Student’s t test was used to determine statistical significance. * Indicates P < 0.05, NS, nonsignificant. Error bars represent SEM.

Article Snippet: ABT-737 and the caspase inhibitor zVAD-FMK were purchased from Selleck Chemicals.

Techniques: Activity Assay, Transfection, Cell Viability Assay, Control, Expressing, Plasmid Preparation, Sterility, Recombinant, Incubation, Staining, Electroporation, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Caspase-3 cleaves and activates the NADase SARM1 to promote apoptosis, linking two cell death mechanisms

doi: 10.1073/pnas.2528118123

Figure Lengend Snippet: Cleavage is required for SARM1 to promote apoptosis. ( A ) Generation of Sarm1 D314A KI mice. Diagram shows the intended SARM1 D314A allele. Sanger sequencing of PCR amplified DNA from Sarm1 D314A KI mice confirmed that D314 was successfully mutated to A . Additional same-sense mutations (marked with *) introduced by the KI strategy ( Methods ) were also confirmed. ( B ) SARM1 WT but not D314A is cleaved during apoptosis. Western blot of SARM1 from DRG cultures under different conditions shows that, unlike WT SARM1, SARM1 D314A is not cleaved. Cultures were treated with anti-NGF/anti-NGF+zVAD for 16 h or ABT-737 (10 µM) for 6 h. Data are representative of three independent experiments. ( C ) Effect of SARM1 D314A KI on primary macrophage apoptosis. Cells were treated with DMSO or TNF (10 ng/mL)+CHX (10 µg/mL) for 7 h. Data are from three biological replicates. ( D ) Effect of SARM1 D314A KI on primary T cell apoptosis. Cells were treated with DMSO or ABT-737 (1 µM) for 19 h. Data are from three biological replicates. ( E ) Effect of SARM1 D314A on DRG Wallerian degeneration. Cultures were imaged 6 h after axotomy. (Scale bar, 50 µm.) Images are representative of three independent experiments. ( F ) Axon degeneration percentage of E was quantified and shown in the bar graphs. Data are from three biological replicates. ( G ) Effect of SARM1 D314A KI on axon degeneration induced by anti-NGF in the presence of caspase inhibitor. Cultures were imaged 20 h after being treated with DMSO, anti-NGF, or anti-NGF+zVAD. (Scale bar, 50 µm.) Images are representative of three independent experiments. ( H ) Axon degeneration percentage of g was quantified and shown in the bar graphs. Data are from three biological replicates. ( I ) Schematic model illustrating caspase-dependent and -independent activation of SARM1. In all panels, Student’s t test was used to determine statistical significance. * Indicates P < 0.05, NS, nonsignificant. Error bars represent SEM.

Article Snippet: ABT-737 and the caspase inhibitor zVAD-FMK were purchased from Selleck Chemicals.

Techniques: Sequencing, Amplification, Western Blot, Activation Assay

Journal: World Journal of Oncology

Article Title: Mitochondria of T Lymphocytes Promote Anti-Pulmonary Tumor Immune Response

doi: 10.14740/wjon1841

Figure Lengend Snippet: Treatment of BH3-mimetic drugs in HCC1195 and HCC1438 cells. (a-c) Cytosol and mitochondrial proteins were extracted from HCC1195, HCC1438, CD3 + T, pulmonary fibroblast cells, and expression of Bcl-2 or Bcl-xL was determined using western blotting. Beta-tubulin was used as a positive marker for the cytosol, while COX IV was used as a positive marker for the mitochondrial fraction. (d) Cell death was measured following treatment with 3 nM ABT199, 19 nM A115463, or 15 µM ABT737 for 1 h, or incubation with 200 pg/µL granzyme B and 100 pg/µL perforin for 2 h. (e, f) The cells were fixed up to 5 h, and apoptosis were determined using ELISA kit. (g) Structures of ABT199 and ABT199-BODIPY. (h) Following treatment of synthetic ABT199-BODIPY, refractive index images of HCC1195 were enlarged at 28, 30, or 32 min, and ABT199-BODIPY were colored in green. (i) Quantified mean fluorescent intensity of the BODIPY signal in cells was measured at 512 nm. Results are the means ± standard error (SE) of six experiments in each group. *Significantly different from cytosol fraction, P < 0.05. # Significantly different from treatment of ABT199-BODIPY at 0 min, P < 0.05. COX IV: cytochrome c oxidase subunit IV; BH3: Bcl-2 homology 3; Bcl-2: B-cell lymphoma 2; Bcl-xL: B-cell lymphoma extra-large; ELISA: enzyme-linked immunoassay; BODIPY: boron dipyrromethene.

Article Snippet: The cells were treated with ABT199 (3 nM, 6960, Tocris, UK), A115463 (19 nM, S7800, Selleck Chemicals, USA), or ABT737 (15 µM, 6835, Tocris, UK) for 1 h. Apoptosis was induced following combinations of recombinant perforin (100 pg/µL, ENZ-PRT313-0010, Enzo Life Science, USA) and granzyme B (200 pg/µL, 10345-H08H, Sinobiological, China).

Techniques: Expressing, Western Blot, Marker, Incubation, Enzyme-linked Immunosorbent Assay, Refractive Index