Journal: Cell Death & Disease

Article Title: Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation

doi: 10.1038/cddis.2013.469

Figure Lengend Snippet: Triptolide induced apoptosis and mitochondrial injury in multiple leukemia cell lines. ( a ) The chemical structure of triptolide, C 20 H 24 O 6 , molecular weight: 360.4. ( b and c ) U937 cells were treated with various triptolide (TPL) concentrations for 24 h or with 40 nM triptolide for different lengths. The percentage of apoptotic cells was determined by FACS analysis using Annexin V/PI staining. Mitochondrial membrane potentials (ΔΨm) were detected by rhodamine-123 staining and flow cytometry. Values represent the mean±S.D. for five separate experiments. Total protein lysates, nuclear extracts, and cytosolic fractions were analyzed by immunoblotting using the indicated antibodies. ( d ) After triptolide treatment, cells were collected and stained with anti-AIF (green) and 4′,6-diamidino-2-phenylindole (DAPI; blue) to identify cellular nuclei. Fluorescence was visualized by a laser confocal scanning microscope. Scale bar represents 10 μ m. These data are representative of three independent experiments. ( e and f ) U937, Jurkat, and HL-60 cells were treated with or without 40 nM triptolide for 24 h, after which apoptosis was determined by FACS analysis using Annexin V/PI staining. Total protein lysates, nuclear extracts, and cytosolic fractions were analyzed by immunoblotting using the indicated antibodies. CF, cleavage fragment; C, cytosolic fractions; N, nuclear extracts

Article Snippet: Human leukemia cell lines U937, Jurkat and HL-60 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Molecular Weight, Staining, Membrane, Flow Cytometry, Western Blot, Fluorescence, Microscopy

Journal: Cell Death & Disease

Article Title: Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation

doi: 10.1038/cddis.2013.469

Figure Lengend Snippet: Triptolide induced Bax translocation in multiple leukemia cell lines. ( a ) U937 cells were treated with various triptolide concentrations for 24 h or with 40 nM triptolide for different lengths, after which cytosolic and mitochondrial fractions were isolated and subjected to immunoblot analysis by using an anti-Bax antibody. ( b ) U937 cells were treated with or without 40 nM triptolide for 24 h. Cells were collected and stained with anti-Bax (green) and MitoTracker (red) to identify mitochondria. Fluorescence was visualized by a laser confocal scanning microscope. Scale bar represents 10 μ m. These data are representative of three independent experiments. ( c ) U937, Jurkat, and HL-60 cells were treated with or without 40 nM triptolide for 24 h, after which cytosolic and mitochondrial fractions were isolated and subjected to immunoblot analysis by using an anti-Bax antibody

Article Snippet: Human leukemia cell lines U937, Jurkat and HL-60 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Translocation Assay, Isolation, Western Blot, Staining, Fluorescence, Microscopy

Journal: Cell Death & Disease

Article Title: Triptolide induces apoptosis in human leukemia cells through caspase-3-mediated ROCK1 activation and MLC phosphorylation

doi: 10.1038/cddis.2013.469

Figure Lengend Snippet: Triptolide induced ROCK1 activation and MLC and MYPT phosphorylation. ( a ) U937 cells were treated with various triptolide concentrations for 24 h or with 40 nM triptolide for different lengths. Total protein lysates were analyzed by immunoblotting using the indicated antibodies. RhoA-GTP was evaluated using a Rhotekin RBD-GST pull-down. ( b ) U937, Jurkat, and HL-60 cells were treated with or without 40 nM triptolide for 24 h. Total protein lysates were analyzed by immunoblotting using the indicated antibodies. Samples from four AML patients ( c ) and two normal donors ( d ) were treated with 40 nM triptolide for 24 or 36 h. The total protein lysates of these samples were obtained and analyzed by immunoblotting using the indicated antibodies

Article Snippet: Human leukemia cell lines U937, Jurkat and HL-60 were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Activation Assay, Phospho-proteomics, Western Blot