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

Journal: Oncogene

Article Title: Star-PAP controls HPV E6 regulation of p53 and sensitizes cells to VP-16

doi: 10.1038/onc.2013.14

Figure Lengend Snippet: Star-PAP controls p53 and E6 expression in high-risk HPV-positive cervical cancer cells downstream of DNA damage. (a) Star-PAP knockdown and VP-16 treatment (50 μM, 6 h) increased p53 and decreased E6 protein levels whereas E6AP levels remained unchanged in HeLa (ATCC), SiHa and CaSki (gifts from Dr Paul F Lambert’s Lab, UW-Madison) cells as analyzed by immunoblotting (IB). Star-PAP small interfering RNA (siRNA) 5′-GUGUGUUUGUCAGUGGCUU-3′; scrambled control siRNA 5′-AGGUAGUGUAAUCGCCUUG-3′. (b) Immunofluorescence (IF) staining demonstrated nuclear accumulation of p53 and depletion of E6 as well as decreased association of the two molecules after Star-PAP knockdown and VP-16 treatment. Scale bar = 10 μm. (c) Cellular protein fractionation and IB showed that the Star-PAP knockdown- and VP-16 treatment-induced increase in p53 levels were restricted in nucleus. Lamin B and β-tubulin were used as controls for nuclear and cytosolic protein fractions, respectively. IB and IF were performed as described.33 Antibodies used: rabbit polyclonal anti-Star-PAP (Anderson’s Lab homemade, Madison, WI, USA);13 mouse monoclonal anti-p53 (Santa Cruz Biotechnology, #sc-126, Santa Cruz, CA, USA); rabbit polyclonal anti-p53 (Santa Cruz Biotechnology, #sc-6243); mouse monoclonal anti-HPV-18 E6 (Santa Cruz Biotechnology, #sc-365089); mouse monoclonal anti-HPV-16 E6 (Santa Cruz Biotechnology, #sc-460); rabbit polyclonal anti-E6AP (Santa Cruz Biotechnology, #sc-25509); mouse monoclonal anti-β-tubulin (Upstate Biotechnology, #05-661, Lake Placid, NY, USA); mouse monoclonal anti-Lamin B (Santa Cruz Biotechnology, #sc-365962); and mouse monoclonal anti-actin (MP Biomedical, #691002, Solon, OH, USA).

Article Snippet: 33 Antibodies used: rabbit polyclonal anti-Star-PAP (Anderson’s Lab homemade, Madison, WI, USA); 13 mouse monoclonal anti-p53 (Santa Cruz Biotechnology, #sc-126, Santa Cruz, CA, USA); rabbit polyclonal anti-p53 (Santa Cruz Biotechnology, #sc-6243); mouse monoclonal anti-HPV-18 E6 (Santa Cruz Biotechnology, #sc-365089); mouse monoclonal anti-HPV-16 E6 (Santa Cruz Biotechnology, #sc-460); rabbit polyclonal anti-E6AP (Santa Cruz Biotechnology, #sc-25509); mouse monoclonal anti-β-tubulin (Upstate Biotechnology, #05-661, Lake Placid, NY, USA); mouse monoclonal anti-Lamin B (Santa Cruz Biotechnology, #sc-365962); and mouse monoclonal anti-actin (MP Biomedical, #691002, Solon, OH, USA).

Techniques: Expressing, Western Blot, Small Interfering RNA, Immunofluorescence, Staining, Fractionation