Journal: PLOS Biology

Article Title: Heme acts as a metabolic brake on erebosis in the Drosophila gut

doi: 10.1371/journal.pbio.3003942

Figure Lengend Snippet: (A) Feeding of CYP P450 inhibitors 1-Aminobenzotriazole (1-ABT) or piperonylbutoxide (PBO) does not restore the inhibition of erebosis by heme. 1-ABT with Ho RNAi and PBO with Mrp5 RNAi result in >50% class0 and no class2, which is similar to Ho RNAi or Mrp5 RNAi with DMSO. (B) ROS levels are not increased by Mrp5 RNAi . Using hsFLP system, Mrp5 RNAi was induced only in GFP+ cells. ROS probe dihydroethidium (DHE, magenta) is not changed in GFP+ Mrp5 RNAi cells (dashed line). (C) Ho RNAi or Mrp5 RNAi does not increase signals of lipid peroxide probe Liperfluo (green), while bleomycin increases them. (D) Feeding of a reducing agent N-acetylcysteine (NAC) does not increase erebosis. P values were calculated using Fisher’s exact test (A, D) and adjusted with the Benjamini–Hochberg method (D). Scale bars, 50 μm.

Article Snippet: Flies were fed with standard food with 15 μM 1-ABT (MedChemExpress HY-103389) or PBO (MedChemExpress HY-B1198) at 30 °C for 7 days.

Techniques: Inhibition

Journal: Drug Delivery

Article Title: Novel reverse electrodialysis-driven iontophoretic system for topical and transdermal delivery of poorly permeable therapeutic agents

doi: 10.1080/10717544.2017.1367975

Figure Lengend Snippet: Mass detector parameters used for the analysis of vitamin C, lopinavir, ketoprofen, and valsartan (IS).

Article Snippet: Lopinavir was purchased from Toronto Research Chemicals Inc. (Toronto, Ontario, Canada).

Techniques:

Journal: Drug Delivery

Article Title: Novel reverse electrodialysis-driven iontophoretic system for topical and transdermal delivery of poorly permeable therapeutic agents

doi: 10.1080/10717544.2017.1367975

Figure Lengend Snippet: In vitro hairless mouse skin permeation profiles of lopinavir after the application of lopinavir-soaked gauze dressing without the RED system (control) and lopinavir-loaded RED system (RED) on the mouse skin fixed in the diffusion cells (A) and the arterial plasma concentration versus time profiles of lopinavir after the transdermal application of lopinavir-soaked gauze dressing without the RED system (control) and lopinavir-loaded RED system (RED) in rats (B). Bullet symbols and their error bars represent the means and standard deviations ( n = 3–4). The asterisk (*) represents a value of the RED group significantly different from that of the control group ( p < .05).

Article Snippet: Lopinavir was purchased from Toronto Research Chemicals Inc. (Toronto, Ontario, Canada).

Techniques: In Vitro, Control, Diffusion-based Assay, Clinical Proteomics, Concentration Assay

Journal: Drug Delivery

Article Title: Novel reverse electrodialysis-driven iontophoretic system for topical and transdermal delivery of poorly permeable therapeutic agents

doi: 10.1080/10717544.2017.1367975

Figure Lengend Snippet: In vitro skin permeation parameters of lopinavir after the application of lopinavir-soaked gauze dressing without the RED system (control) and lopinavir-loaded RED system (RED) on the hairless mouse skin fixed in the diffusion cells ( n = 3–4).

Article Snippet: Lopinavir was purchased from Toronto Research Chemicals Inc. (Toronto, Ontario, Canada).

Techniques: In Vitro, Control, Diffusion-based Assay

Journal: Drug Delivery

Article Title: Novel reverse electrodialysis-driven iontophoretic system for topical and transdermal delivery of poorly permeable therapeutic agents

doi: 10.1080/10717544.2017.1367975

Figure Lengend Snippet: Pharmacokinetic parameters of lopinavir after the transdermal application of lopinavir-soaked gauze dressing without the RED system (control) and lopinavir-loaded RED system (RED) in rats ( n = 3–4).

Article Snippet: Lopinavir was purchased from Toronto Research Chemicals Inc. (Toronto, Ontario, Canada).

Techniques: Control

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a-c , NINJ1 oligomerization (Native-PAGE) and LDH-release from WT and NINJ1 KO iPSDMs pretreated with glycine or inhibitors as indicated and infected with Mtb mc²6206, MOI 20 for 4h ( a, b ), Mtb H37Rv, 7.5 MOI for 24h ( c ), or treated with cell death stimuli for 4h: d , pyroptosis – LPS and nigericin (LPS+N) w/wo NLRP3 inhibitor MCC950; e , apoptosis – venetoclax; f , necroptosis – Z-VAD-FMK+BV-6+TNFa (zBT) w/wo RIPK3 inhibitor GSK’872; g , ferroptosis – RSL-3 w/wo Ferrostatin-1. Data are means (bars) ± s.e.m. of at least three independent experiments (circles), each with three replicates per condition. *P<0,05; **P<0,01; ****P<0,0001 by RM two-way Anova with Tukey’s multiple comparisons test of treatments within each cell line and stimuli between cell lines. For gel source data, see Supplementary Figure 1.

Article Snippet: Apoptosis was induced with the BCL2-inhibitor, Venetoclax (125 μM, TOCRIS, 6960/10) for 4 hours.

Techniques: Clear Native PAGE, Infection

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a, Lysates of WT, NINJ1 KO and GSDMD KO iPSC-derived monocytes (m) and macrophages (M) were subjected to SDS-PAGE and immunoblotted for NINJ1, GSDMD and β-actin. A3 and C6 are NINJ1 KO clones; A7, B8 are GSDMD KO clones. b, Human monocyte-derived macrophages were transfected with siRNA targeting NINJ1 or a non-targeted control and infected with Mtb mc²6206, MOI 20, for 4h. Shown is LDH-release and NINJ1 expression level by qPCR. Data are presented as mean ± s.e.m of four donors (shown with individual symbols). *P<0,05; **P<0,01; ****P<0,0001 by RM two-way Anova with uncorrected Fisher’s LSD. c-j, LDH-release from iPSDM WTs and NINJ1 KO clones A3, C6 ( c-f ) or GSDMD KO clones A7, B8 ( g-j ) pretreated with glycine or inhibitors as indicated and treated with cell death stimuli for 4h: pyroptosis – LPS and nigericin (LPS+N) w/wo NLRP3 inhibitor MCC950 ( c, g ); apoptosis – venetoclax ( d, h ); necroptosis – Z-VAD-FMK+BV-6+TNFα (zBT) w/wo RIPK3 inhibitor GSK’872 ( e, i ); ferroptosis – RSL-3 w/wo Ferrostatin-1 ( f, j ). ( c-f ) Data are means (bars) ± s.e.m. of three (A3) or two (C6) independent experiments (circles), each with three replicates per condition. *P<0,05; **P<0,01; ****P<0,0001 by RM two-way Anova with Tukey’s multiple comparisons test. ( g-j ) Data are means (bars) ± s.d. of two or three technical replicates (circles) from 1 independent experiment.

Article Snippet: Apoptosis was induced with the BCL2-inhibitor, Venetoclax (125 μM, TOCRIS, 6960/10) for 4 hours.

Techniques: Derivative Assay, SDS Page, Clone Assay, Transfection, Control, Infection, Expressing

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a-d , Cytokine release from WT iPSDMs pretreated with glycine or inhibitors as indicated for 30 min and treated with cell death stimuli for 4h: pyroptosis – LPS and nigericin (LPS+N) w/wo NLRP3 inhibitor MCC950 ( a ); apoptosis – venetoclax ( b ); necroptosis – zVAD+BV-6+TNFα (zBT) w/wo RIPK3 inhibitor GSK’872 ( c ); ferroptosis – RSL-3 w/wo Ferrostatin-1 ( d ). Cytokine levels were assessed by multiplex ELISA. Data are means (bars) of two individual experiments (circles), each with two replicates.

Article Snippet: Apoptosis was induced with the BCL2-inhibitor, Venetoclax (125 μM, TOCRIS, 6960/10) for 4 hours.

Techniques: Multiplex Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a , Representative images of NINJ1 KO THP-1 cells expressing hNINJ1-mNG (THP1-NINJ1-mNeonGreen) pretreated with glycine ( a , right column) before cell death stimuli venetoclax (apoptosis), LPS + nigericin (LPS+N, pyroptosis), RSL-3 (ferroptosis) for 4h, or infection with Mtb mc26206-BFP for 20h. Time-lapse TIRF (mNG), widefield (DRAQ7 and BFP) and brightfield microscopy images of NINJ1-mNG (green), DRAQ7 (red) and Mtb (blue), and morphology, respectively. Scale bars 10 mm. b , Quantification of NINJ1-mNG puncta densities at 4h (LPS+N, RSL-3) or 20h (Mtb) as represented in ( a ). Data are means (bars) ± s.e.m from 1-4 independent experiments (circles), each including analysis of 4-15 cells per condition. Significant differences are indicated as *P<0,05 by RM two-way Anova with Tukey’s multiple comparisons test. c , Representative time-lapse images of NINJ1 clustering (TIRF, green) and DRAQ7 uptake (red) upon Mtb mc26206-BFP infection (blue) temporally aligned by start of NINJ1 clustering (t=0). Time in min. Scale bars 10 mm. d, e , Fitted curves ( d ) and t50 values (x value where y=50% max.) of sigmoidal fits ( e ) of NINJ1 oligomerization kinetics during Mtb-infection, apoptosis (venetoclax), pyroptosis (LPS+N) or ferroptosis (RSL-3) monitored with frame rates of 8-10 sec (Venetoclax, RSL-3, LPS+N) or 5 min (Mtb-infection). 3-5 cells analyzed per condition. Individual kinetic curves and representative images for RCD induction are shown in Extended Data Figure 3.

Article Snippet: Apoptosis was induced with the BCL2-inhibitor, Venetoclax (125 μM, TOCRIS, 6960/10) for 4 hours.

Techniques: Expressing, Infection, Microscopy

Journal: bioRxiv

Article Title: NINJ1 is activated by Mycobacterium tuberculosis ESX-1 secreted effector EsxA and mediates necrosis of infected human macrophages

doi: 10.1101/2025.11.04.685750

Figure Lengend Snippet: a, b, Quantification of NINJ1-mNG puncta densities monitored by TIRF microscopy ( a ) and phagocytosed bacteria monitored by intracellular BFP intensities ( b ) before NINJ1 inhibition by glycine and after 4h of RCD induction (Venetoclax, LPS+N, RSL-3) or after 3h and 20h of Mtb mc²6206-BFP infection. Data are means (bars) ± s.e.m from 1-4 independent experiments (circles), each including analysis of 4-15 cells per condition. Significant differences are indicated as *P<0,05 by RM two-way Anova with Tukey’s multiple comparisons test. c,d, Kinetics of NINJ1 puncta densities and DRAQ7 intensities monitored with frame rates of 8-10 sec (Venetoclax, RSL-3, LPS+N) with (c) representative Time-lapse images of NINJ1 clustering (TIRF, green) and DRAQ7 (WF, red) aligned by start of NINJ1 clustering. Time in sec. Scale bars 10 μm. Intensity values were normalized to the highest value for each cell and aligned by the start of NINJ1 oligomerization. Data are means ± s.d. for 3-5 cells per condition with sigmoidal fits for NINJ1-mNG puncta density increase (green) and mono-exponential increase for DRAQ7 intensities (red).

Article Snippet: Apoptosis was induced with the BCL2-inhibitor, Venetoclax (125 μM, TOCRIS, 6960/10) for 4 hours.

Techniques: Microscopy, Bacteria, Inhibition, Infection

Journal: Cell Death Discovery

Article Title: 5-Iodotubercidin sensitizes cells to RIPK1-dependent necroptosis by interfering with NFκB signaling

doi: 10.1038/s41420-023-01576-x

Figure Lengend Snippet: A MK2-deficient MEFs transduced with MK2 expression or control vector were treated with different doses of 5-ITu in the presence or absence of TNF (10 ng/ml) for 6 h. B Cells of the indicated genotype were treated with 10 µM 5-ITu for 6 and 24 h and cell viability was quantified. C MK2-deficient MEFs transduced with MK2 expression or control vector were treated with indicated small molecules (5 µM ABT-702, 100 nM gemcitabine, 5 µM etoposid, 5 µM doxorubicin and 5 µM staurosporine) in the presence or absence of TNF for 6 h and cell viability was assessed. D MK2-deficient MEFs transduced with MK2 expression or control vector were treated as indicated and viability was quantified after 6 h treatment. E , F RAW264.7 cells were treated as indicated to monitor the effect of 5-ITu in LPS-induced necroptosis ( E ) and SM-mediated autocrine TNF-dependent death ( F ). Average values of n = 3 independent wells are plotted ± SD (** p ≤ 0.001, *** p ≤ 0.0001, **** p ≤ 0.0001).

Article Snippet: The following reagents were used for cell treatments at given concentrations: LPS ( Escherichia coli O55:B5, #L2880, Sigma-Aldrich, 100 ng ml −1 ), recombinant human TNFα (rHuTNF, #50435.50, Biomol, 10 ng/ml), Birinapant/SM (#HY-16591, MedChem Express, 1 μM), pan-caspase inhibitor zVAD-fmk (#4026865.0005, Bachem, 25 μM), MK2 inhibitor PF3644022 (#4279, Tocris, 5 μM), RIPK1 inhibitor Nec-1 (#BML-AP309-0020, Enzo Life Sciences, 50 μM), RIPK1 inhibitor Nec-1s (# 10-4544-5 mg, Tebu-Bio, 50 μM), RIPK3 inhibitor GSK872 (#HY-101872, MedChem Express, 5 μM), IKKβ inhibitor BMS345541 (#Axon 1731, Axon Medchem, 5 μM), TBK1/IKKε inhibitor BX795 (#T1830, Tebu-Bio), 5-Iodotubercidin (#HY-15424, MedChem Express, 2.5–10 μM), ABT-702 dihydrochloride (#HY-103161, MedChem Express, 5 μM), Staurosporine (#81590, Cayman, 5 μM), Etoposide (#E1383, Sigma, 5 μM), Doxorubicin (#15007, Cayman, 5 μM), Gemcitabine (#S1714, Selleckchem, 100 nM).

Techniques: Transduction, Expressing, Control, Plasmid Preparation

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: Aging (Albany NY)

Article Title: Transient metabolic improvement in obese mice treated with navitoclax or dasatinib/quercetin

doi: 10.18632/aging.103607

Figure Lengend Snippet: Senolytic treatment strategy. ( A ) Scheme of the senolytic treatments strategy: C57BL/6HsdOla male mice of 12 weeks of age were fed on 45% HFD for 4 months. Then, 5 cycles of senolytic treatment and glucose homeostasis assessment were undertaken: first, mice were treated daily by oral gavage with the senolytic or its vehicle for 5 consecutive days, and rested for 1 week. The third week of each cycle, an insulin tolerance test (I) and a glucose tolerance test (G) were performed. After the fifth cycle was finished, mice were treated again with senolytics for 5 consecutive days by oral gavage, and sacrificed at the fifth day of treatment (Sac). ( B ) Mouse body weight at the first day of every senolytic treatment, indicated for each of the 5 cycles. ( C ) Food intake recorded at cycles 1 and 2. ( D ) Blood samples were collected every fifth day of each cycle of senolytic treatments, right after the last oral administration, and platelet counts were determined. V-Nav: vehicle for navitoclax. Nav: navitoclax. V-D/Q: vehicle for dasatinib/quercetin. D/Q: dasatinib/quercetin. Bars and dots represent the average of the indicated number of mice. Error bars represent the standard error of the mean (s.e.m.). Statistical significance was assessed using the two-way ANOVA test with Sidak’s correction for multiple comparisons ( B ); the one-way ANOVA test with Tukey’s correction for multiple comparisons ( C ); or the unpaired two-tailed Student's t-test ( D ). **, p<0.01; ***, p<0.001.

Article Snippet: At the beginning of each cycle, mice were weighed (this body weight record is shown at ) and treated by oral gavage with either navitoclax (MedChemExpress, HY-10087; 50 mg/kg dissolved in 15% DMSO and 15% PEG 400); with a mixture of dasatinib (MedChemExpress, HY-10181; 5 mg/kg dissolved in 10% PEG 400), and quercetin (MedChemExpress, HY-18085; 50 mg/kg dissolved in 10% PEG 400); or with their corresponding vehicles (15% DMSO and 15% PEG 400 for navitoclax, or 10% PEG 400 for D/Q).

Techniques: Two Tailed Test

Journal: Aging (Albany NY)

Article Title: Transient metabolic improvement in obese mice treated with navitoclax or dasatinib/quercetin

doi: 10.18632/aging.103607

Figure Lengend Snippet: Senescence markers in DIO-mice after senolytic treatment. ( A ) Macroscopic view of perigonadal white adipose tissue (pWAT) from mice treated with the 5 cycles of senolytics and stained for senescence-associated β-Galactosidase (SA-β-Gal) activity. V: vehicle. Nav: navitoclax. D/Q:dasatinib/quercetin. ( B ) Microscopic images of the same pWATs shown in ( A ). ( C ) Quantification of the blue area, positive for SA-βGal, of 6 fields per condition from the pWATs shown in ( A ) and ( B ). ( D ) mRNA expression analysis of the indicated genes in pWAT obtained at the day of sacrifice. Statistical significance was assessed using the one-way ANOVA test with Tukey's correction for multiple comparisons ( C ) or the two-tailed unpaired Student's t-test between each treatment and its control ( D ) Asterisks refer to the comparison with the corresponding vehicle-treated mice. *, p<0.05; **, p<0.01.

Article Snippet: At the beginning of each cycle, mice were weighed (this body weight record is shown at ) and treated by oral gavage with either navitoclax (MedChemExpress, HY-10087; 50 mg/kg dissolved in 15% DMSO and 15% PEG 400); with a mixture of dasatinib (MedChemExpress, HY-10181; 5 mg/kg dissolved in 10% PEG 400), and quercetin (MedChemExpress, HY-18085; 50 mg/kg dissolved in 10% PEG 400); or with their corresponding vehicles (15% DMSO and 15% PEG 400 for navitoclax, or 10% PEG 400 for D/Q).

Techniques: Staining, Activity Assay, Expressing, Two Tailed Test, Control, Comparison

Journal: Cell reports

Article Title: Poly(ADP-ribosyl)ation of TIMELESS limits DNA replication stress and promotes stalled fork protection

doi: 10.1016/j.celrep.2024.113845

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Veliparib , MedChemExpress , Cat#HY-10129.

Techniques: Control, Virus, Recombinant, TNKS1 Histone Ribosylation Assay, Transfection, Protease Inhibitor, In Situ, DNA Extraction, Mutagenesis, Cell Viability Assay, Staining, Negative Control, Software

Journal: NPJ Precision Oncology

Article Title: Microfluidic-based dynamic BH3 profiling predicts anticancer treatment efficacy

doi: 10.1038/s41698-022-00333-0

Figure Lengend Snippet: a FACS-based DBP curves after incubation for 16 h with DMSO, imatinib, ripretinib, and dactolisib in primary cancer cells. Results are represented as the percentage of cells with cytochrome c retained inside the mitochondria after incubation with increasing concentrations of BIM peptide. b Quantification of % priming (percentage of cells with cytochrome c scape) in primary cancer cells after incubation with BIM, BAD, HRK, and MS1 peptides. c Quantification of % priming after BIM peptide incubation in primary cancer cells incubated for 16 h with DMSO, dactolisib, and the combination of dactolisib and venetoclax. d Visualization of primary cancer cells seeded inside the cell chambers of the microfluidic chip after treatment with DMSO, dactolisib and the combination of dactolisib and venetoclax for 16 h and exposure to increasing concentrations of BIM peptide generated using microfluidics. Alive cells at the start of the assay are marked in green (calcein AM) and TMRE (red field) is used to identify initiation of MOMP (marking the start of apoptosis). Scale bars, 100 μm. e Microfluidic-based DBP curves after incubation of the primary cancer cells for 16 h with DMSO, dactolisib, and the combination of dactolisib and venetoclax. Results are represented as the percentage of cells with positive TMRE signal after incubation with increasing concentrations of BIM peptide produced by the microfluidic gradient generator. f Quantification of % priming (percentage of cells with negative TMRE signal) in primary cancer cells inside the microfluidic chip. Values were chosen in the BIM peptide condition where the DMSO-treated cells started to engage MOMP and lose TMRE staining.

Article Snippet: Imatinib, dactolisib, and gefitinib were obtained from LC Laboratories (Woubourn, MA, USA); venetoclax was purchased at MedChemExpress (Monmouth Junction, NJ, USA), and repretinib from Selleckchem (Munich, Germany).

Techniques: Incubation, Generated, Produced, Staining