Journal: Clinical and Translational Medicine

Article Title: IRF4 contributes to chemoresistance in IGH::BCL2‐positive diffuse large B‐cell lymphomas by mediating BCL2‐induced SOX9 expression

doi: 10.1002/ctm2.70336

Figure Lengend Snippet: Silencing BCL2 suppresses sex‐determining region Y (SRY)‐box 9 protein (SOX9) expression in BCL2 overexpressed germinal centre B‐cell‐like (GCB) diffuse large B‐cell lymphoma (DLBCL) subsets. (A and B) Immunoblotting and real‐time polymerase chain reaction (PCR) assay of BCL2 and SOX9 protein and mRNA levels in a panel of DLBCL cell lines (Karpas‐422, DB, SUDHL2 and SUDHL8), DB cells were transduced either with lentiviral encoding scramble control, shBCL2#1 or shBCL2#2 plasmids for 72 h, respectively, prior to subject flow cytometry sorting of Green fluorescent protein (GFP)‐positive cells to generate stable transfectants. (C and D) Immunoblotting or real‐time PCR assays of protein or mRNA level of BCL2 and SOX9. Two‐way analysis of variance (ANOVA) was used to compare scramble Ctrl to shSOX9#1 and shSOX9#2. (E and F) Immunoblotting assay of BCL2 and SOX9 protein levels in Karpas‐422 and DB cells were either treated with indicated doses of ABT‐737 or ABT‐199 for 48 h. (G and H) Real‐time PCR of BCL2 and SOX9 mRNA levels in Karpas‐422 and DB cells were either treated with 2.5 µM ABT‐737 or ABT‐199 for 48 h. Two‐way ANOVA was performed to compare vehicle Ctrl to treated. (I) BCL2 and SOX9 immunostainings were denoted as (+), (++) and (+++) to indicate the expression levels of BCL2 and SOX9. Negative control was included to eliminate the false‐positive staining. Positivity of BCL2 or SOX9 immunostaining was measured using ImageJ software, the simple linear regression statistical analysis was carried out to determine the association between SOX9 and BCL2 positives ( n = 6, 200× magnification). β‐Tubulin or GAPDH was included as indications of equal loading. Protein levels were quantified (normalised to housekeeping genes) using ImageJ software and graph was generated using GraphPad version 9.0. All experiments were repeated three times, and graph with error bars show the data represent the mean ± standard deviation (SD) from technical triplicates ( **** p < .005).

Article Snippet: Doxorubicin (catalog no. HY‐15142), ABT‐199 (catalog no. HY‐15531), ABT‐737 (catalog no. HY‐50907), cyclophosphamide (catalog no. HY‐17420), vincristine (catalog no. HY‐N0488A), prednisone (catalog no. HY‐B0214) and rituximab (catalog no. HY‐P9913) were purchased from MedChem Express (Monmouth Junction).

Techniques: Expressing, Western Blot, Real-time Polymerase Chain Reaction, Control, Flow Cytometry, Negative Control, Staining, Immunostaining, Software, Generated, Standard Deviation

Journal: Clinical and Translational Medicine

Article Title: IRF4 contributes to chemoresistance in IGH::BCL2‐positive diffuse large B‐cell lymphomas by mediating BCL2‐induced SOX9 expression

doi: 10.1002/ctm2.70336

Figure Lengend Snippet: Sex‐determining region Y (SRY)‐box 9 protein (SOX9) reduced drug sensitivity of BCL2 overexpressed germinal centre B‐cell‐like (GCB) diffuse large B‐cell lymphoma (DLBCL) subsets. (A‒C) Karpas‐422, DB (SOX9 high expressing cells) or SUDHL6, OCI‐LY1 (SOX9 low expressing cells) were treated either with vehicle control, 2.5 µM doxorubicin, 2.5 µM ABT‐199 or various doses of ABT‐737 (.5, 1.5 and 2.5 µM) for 48 h, respectively, followed by flow cytometry to determine the percentage of viable cells. Two‐way analysis of variance (ANOVA) was performed to compare the difference of viable cells (%) between SOX9 high expressing cells and SOX9 low expressing cells upon drug treatment. (D‒F) Karpas‐422 or DB cells were transduced either with scramble control, shSOX9#1 or shSOX9#2 lentiviral plasmids for 72 h, followed by flow cytometry sorting out GFP‐positive cells to obtain stable transfectants. Stable cells were then treated either with vehicle control, 2.5 µM DOX, 2.5 µM ABT‐199 or ABT‐737 (.5, 1.5 and 2.5 µM) for 48 h, followed by flow cytometry to determine the percentage of viable cells. Two‐way ANOVA was performed to compare the difference of viable cells (%) between scramble Ctrl and SOX9 silencing groups. (G and H) OCI‐LY1 cells were transduced either with empty vector or PCDH‐SOX9 overexpression lentiviral plasmids for 48 h followed by flow sorting of GFP‐positive cells to obtain stable transfectants. SOX9‐overexpressing OCI‐LY1 stable transfectants were then either with vehicle control, 2.5 µM doxorubicin or 2.5 µM ABT‐199 for 48 h, followed by flow cytometry to determine the percentage of viable cells. One‐way ANOVA was performed to compare the difference between empty vector and SOX9 overexpression groups. (I) Ten millions of lentiviral encoding scramble control, shSOX9#1 and shSOX9#2 plasmids transducing BD cells were engrafted to generate DLBCL xenografts via subcutaneous injection prior to administrate mice either with vehicle control or DOX (2.5 mg/kg) every 2 days for four times or ABT‐199 (25 mg/kg) very 2 days for three times, mice were sacrificed prior to subject to tumourigenesis measurements. Tumour size (I), the percentage of TUNEL‐positive cells (J) or the percentage of Ki67‐positive cells (K) were assessed. Percentage of viable cells by flow cytometry represents the data normalised to vehicle Ctrl. All experiments were repeated three times, and graph with error bars show the data represent the mean ± standard deviation (SD) from technical triplicates ( * p < .05; ** p < .01; *** p < .001).

Article Snippet: Doxorubicin (catalog no. HY‐15142), ABT‐199 (catalog no. HY‐15531), ABT‐737 (catalog no. HY‐50907), cyclophosphamide (catalog no. HY‐17420), vincristine (catalog no. HY‐N0488A), prednisone (catalog no. HY‐B0214) and rituximab (catalog no. HY‐P9913) were purchased from MedChem Express (Monmouth Junction).

Techniques: Expressing, Control, Flow Cytometry, Plasmid Preparation, Over Expression, Injection, TUNEL Assay, Standard Deviation

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: Nucleic Acids Research

Article Title: Reduction of lamin B receptor levels by miR-340-5p disrupts chromatin, promotes cell senescence and enhances senolysis

doi: 10.1093/nar/gkab538

Figure Lengend Snippet: miR-340-5p sensitizes pre-senescent fibroblasts to senolytic death. (A, B) WI-38 fibroblasts (PDL20) were exposed to IR and transfected with miR Ctrl, miR-340-5p or anti-miR-340-5p and cultured for additional 3 days. Cells were then treated with ABT-737 (10 μM) or with the combination of dasatinib (20 μM) + quercetin (8 μM) (D + Q) for 24 h. Cell viability (A) and caspase 3/7 activity (B) were quantified and plotted. (C, D) WI-38 fibroblasts (PDL20) were exposed to IR and transfected with siCtrl alone or siLBR alone or with anti-miR-340-5p and cultured for additional 3 days. Cells were then treated with ABT-737 or the combination D + Q [as in panel (A)] for 24 h. Cell viability (C) and caspase 3/7 activity (D) were quantified and plotted. (E) Schematic illustrating the proposed sensitization of pre-senescent cells to senolytic drugs by miR-340-5p and the proposed protection against senolytics by anti-miR-340-5p. Data in panels (A)–(D) represent the mean values ± SD from three biological replicates. Significance was established using Student’s t-test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001.

Article Snippet: Cells were transfected as described and treated with senolytics [ABT-737 10 μM (Tocris); dasatinib 20 μM + quercetin 8 μM (Selleckchem)] for 24 h. Viability was monitored by cell counting and represented as the percentage of remaining cells compared to the number of cells at the start of the experiment.

Techniques: Transfection, Cell Culture, Activity Assay