flow cytometric analyses  (GraphPad Software Inc)

Journal: Cell Death Discovery

Article Title: Novel meriolin derivatives activate the mitochondrial apoptosis pathway in the presence of antiapoptotic Bcl-2

doi: 10.1038/s41420-024-01901-y

Figure Lengend Snippet: A – C About 5 × 10 5 Ramos lymphoma or Jurkat leukemia cells were treated with increasing concentrations of meriolin 16 ( A ), 31 ( B ), and 36 ( C ) or 2.5 µM staurosporine (STS), either alone or in combination (pre- and cotreatment) with the pan-caspase inhibitor QVD (10 µM). After 24 h of incubation, apoptosis-related DNA degradation was detected via flow-cytometric measurement of propidium iodide-stained apoptotic hypodiploid nuclei . Error bars = SD of triplicates. D – F About 5 × 10 5 Ramos or Jurkat cells were treated with 1 or 10 µM of meriolin 16 ( D ), 31 ( E ), and 36 ( F ) or 2.5 µM staurosporine (STS) for up to 8 h. Subsequently, caspase-3 activity was determined by measurement of the fluorescence of the profluorescent caspase-3 substrate DEVD-AMC in a spectrofluorometer. Error bars = SD of triplicates. G – I About 1 × 10 6 Ramos or Jurkat cells were treated with 1 µM of meriolin 16 ( G ), 31 ( H ), and 36 ( I ), 0.1% (v/v) DMSO (solvent control), or 2.5 µM staurosporine (STS) for the indicated time periods, either alone or in combination (pre- and cotreatment) with the pan-caspase inhibitor QVD (10 µM). Cleavage of the caspase substrate PARP was detected by immunoblotting. Solid arrowheads indicate the uncleaved form of PARP (p116); open arrowheads indicate the cleaved form (p85). Immunoblotting for tubulin or β-actin was used as a loading control.

Article Snippet: All flow-cytometric analyses were performed on an LSR-Fortessa™ (Becton Dickinson, Heidelberg, Germany).

Techniques: Incubation, Staining, Activity Assay, Fluorescence, Solvent, Western Blot

Journal: Cell Death Discovery

Article Title: Novel meriolin derivatives activate the mitochondrial apoptosis pathway in the presence of antiapoptotic Bcl-2

doi: 10.1038/s41420-024-01901-y

Figure Lengend Snippet: A , B Meriolins do not induce apoptosis via the death receptor pathway. A About 5 × 10 5 caspase-8 proficient Jurkat cells (Jurkat-Casp8-pos., black bars) or caspase-8 deficient Jurkat cells (Jurkat-Casp8-neg., white bars) were treated with 0.1% (v/v) DMSO (solvent control), 1 µM of meriolin 16, 31, and 36, 50 µM of etoposide (Eto), 2.5 µM staurosporine (STS), or the death receptor ligand TRAIL (40 ng/ml; positive control). After 24 h, apoptosis was assessed by propidium iodide staining of apoptotic hypodiploid nuclei and flow cytometry. B About 1 × 10 6 caspase-8 proficient Jurkat cells (Jurkat-Casp8-pos.) or caspase-8 deficient Jurkat cells (Jurkat-Casp8-neg.) were treated as in (A) for the indicated time period. Cleavage of the caspase substrate PARP was detected by immunoblotting. Solid arrowheads indicate the uncleaved form of PARP (p116); open arrowheads indicate the cleaved form (p85). C , D Meriolin 16, 31, and 36 induce apoptosis in the presence of antiapoptotic Bcl-2. Jurkat cells stably transfected with vectors encoding Bcl-2 (Jurkat Bcl-2; black bars) or empty vector (Jurkat vector; white bars) were treated with 0.1% (v/v) DMSO, 1 µM of meriolin 16, 31, and 36, 50 µM etoposide (Eto), or 2.5 µM staurosporine (STS). C After 24 h, apoptosis was assessed by flow-cytometric measurement of apoptotic hypodiploid nuclei. D After the indicated time period, cleavage of the caspase substrate PARP was detected by immunoblotting. E , F Meriolin-induced apoptosis requires caspase-9. Caspase-9 deficient (Jurkat Casp9-neg., white bars) or caspase-9 proficient Jurkat cells (Jurkat Casp9-pos., black bars) were treated with 0.1% (v/v) DMSO, 1 µM of meriolin 16, 31, and 36, 50 µM etoposide (Eto), or 2.5 µM staurosporine (STS). E After 24 h, apoptosis was assessed by flow-cytometric measurement of apoptotic hypodiploid nuclei. F After the indicated time period, cleavage of the caspase substrate PARP was detected by immunoblotting. Immunoblot of expression of caspase-9 is also shown. B , D , F Immunoblotting for GAPDH or tubulin was used as loading control.

Article Snippet: All flow-cytometric analyses were performed on an LSR-Fortessa™ (Becton Dickinson, Heidelberg, Germany).

Techniques: Solvent, Positive Control, Staining, Flow Cytometry, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Expressing

Journal: Cell Death Discovery

Article Title: Novel meriolin derivatives activate the mitochondrial apoptosis pathway in the presence of antiapoptotic Bcl-2

doi: 10.1038/s41420-024-01901-y

Figure Lengend Snippet: A Monitoring of the mitochondrial membrane potential (ΔΨm) of Ramos and Jurkat cells upon addition of 10 µM of meriolin 16, 31, or 36, 0.1% (v/v) DMSO (diluent control) or 10 µM CCCP (mitochondrial uncoupler, positive control) by flow-cytometric measurement of TMRE fluorescence. B The kinetics of the cleavage of the long isoforms (L1,2) of the dynamin-like GTPase OPA1 was determined by immunoblotting in Ramos (left panel) and Jurkat cells (right panel). Cells were treated as in ( A ) for the indicated time points. Immunoblotting for tubulin was used as a loading control. C Meriolin 16, 31, and 36 induce mitochondrial fragmentation (fission) in HeLa cells, stably expressing the fluorescent dye mito-DsRed targeted to the outer mitochondrial membrane. Cells were treated with DMSO (0.1% v/v), 1 µM of meriolin 16, 31, or 36, or 10 µM CCCP (positive control) for the indicated time points and mitochondrial morphology was assessed by microscopy (Apotome, Zeiss Axiovert). Shown are representative images.

Article Snippet: All flow-cytometric analyses were performed on an LSR-Fortessa™ (Becton Dickinson, Heidelberg, Germany).

Techniques: Membrane, Positive Control, Fluorescence, Western Blot, Stable Transfection, Expressing, Microscopy