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  • 99
    Millipore camptothecin
    Insig2 inhibits apoptosis. ( a ) Three HCT116-Insig2-Myc clones and mock transfected HCT116 cells were cultured overnight, treated with 5 μM 5-FU and 50 ng/ml anti-Fas, or 1 μM <t>camptothecin</t> for 24 hr. Both floating and attached cells were
    Camptothecin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3488 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Selleck Chemicals camptothecin
    High-throughput assessment of fractional killing between conditions. (A,B) Assessment of live cell counts (mKate2 positive [mKate2 + ] objects) over lethal compound concentration-response series in two cell lines. Measurements were performed at 72 h. Btz: bortezomib, Vinb: vinblastine. Percent reduction in live cell counts between different compound concentrations is indicated by black lines and arrows and discussed in the main text. (C,D). Absolute metabolic viability measurements at select dose points. Colored circles in C and D correspond to specific doses indicated on the curves in A and B, respectively. (E,F). Metabolic viability measurements normalized per cell. Colored circles in E and F correspond to specific doses indicated on the curves in A and B, respectively. (G) Heatmap summaries of normalized, log 10 -scaled live mKate2 positive (mKate2+) live cell counts, SYTOX Green positive (SG+) dead cell counts, and integrated lethal fraction (Let. Frac.) scores over time (x-axis) and concentration (y-axis) for ten compounds in T98G N cells. For the mKate2 + heatmaps, live cell counts were normalized to 1 at time = 0 and expressed on a log scale. Values more than 10-fold lower than the starting values are colored black. Etop: etoposide, Cpt: <t>camptothecin,</t> Sts: staurosporine, Thap: thapsigargin, Tun: tunicamycin, Era: erastin, Pac, paclitaxel. (H) Live and dead cell counts for T98G N cells, extracted from the data shown in G. Data are from three independent experiments and represent mean ± SD (A-F, H) or mean (G).
    Camptothecin, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 92/100, based on 104 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Tocris camptothecin
    Effects of A. argyi extract on the function of T lymphocytes. For (A , B) , lymphocytes (2 × 10 5 ) were left unstimulated (unstim.) or were activated with anti-CD3 and anti-CD28 mAbs (100 ng each and incubated for 24 h with medium (unstim., stim.), cyclosporine A (CsA; 4.16 µM), <t>camptothecin</t> (CPT; 300 µM), or the A. argyi extract. Cells were stained with anti-CD69-FITC and anti-CD4-APC (A) or anti-CD25-PE and anti-CD4-APC (B) . Expression of surface markers (CD69, CD25, CD4) was analyzed by flow cytometry, and the amounts of activated CD4 + and CD4 – T lymphocytes were determined. For (C , D) expanded lymphocytes (2 × 10 5 ) were incubated for 20 h with medium (unstim., stim.), cyclosporine A (CsA; 4.16 µM), camptothecin (CPT; 300 µM), parthenolide (20 µM) or the A. argyi extract. Subsequently, the cells were stimulated with PMA/Ionomycin for 4 h at 37°C. IL-2 (C) and IFN-γ (D) were quantified in the supernatants by ELISA. The results of three different experiments are summarized and are depicted as mean ± SD in relation to the untreated, stimulated control. *p
    Camptothecin, supplied by Tocris, used in various techniques. Bioz Stars score: 96/100, based on 51 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc camptothecin
    Effects of siRNAs specific to CXXC5 on cellular growth. MCF7 cells grown in 10% CD-FBS containing medium for 48 h were transiently transfected without (UT) or with 10 nM CtS or siRNA#10 in the absence (0.01% ethanol) or the presence of 10 −8 M E2 up to 72 h. ( a ) Cells were subjected to cell counting using a hemocytometer. Results, as the mean ± S.E. of three independent determinations, depict fold change in cellular growth compared with those observed with cells at time 0 in the absence of E2, which is set to 1. In ( a–f ) the asterisk with a superscript “a” indicates significant difference from the corresponding ethanol-treated group (-E2); whereas superscript “b” depicts a significant difference from CtS transfected cells in the presence of E2. ( b ) The cDNA library generated from total RNA (50 ng) isolated from transfected cells for 48 h was subjected to qPCR using a primer set specific to CXXC5. ( c ) Nuclear extracts from untransfected (UT) or transfected cells were subjected to WB using an antibody for CXXC5, ERα or HDAC1. NS denotes non-specific protein; Molecular mass in KDa is indicated. Transfected cells were subjected to cell counting ( d ) and MTT assay ( e ). In ( b , d , e) , results depicting the mean ± S.E. of three independent determinations indicate fold change in mRNA levels ( b ) or cell numbers ( d , e ) compared with CtS in the absence of E2, which is set to 1. Transfected MCF7 cells were subjected to flow cytometry ( f ) with results depicting the percent of cells in the G1, G2 and S phases, or to Annexin V assay followed by flow cytometry ( g ). In ( f , g ), results are the mean ± SEM of three independent experiments. ( h i ) Untransfected (UT) or transfected cells were also subjected to the cell death inducer <t>camptothecin</t> (2 nM) for 24 h without (−E2) or with E2 (E2). Cells were then subjected to Annexin V assay ( h ) with results as the mean ± S.E. of three independent experiments depicting percent change in apoptotic (upper right quadrant) and death (upper left quadrant) cells, or to WB ( i ) using a PARP1-specific antibody. P and CP denote the uncut and cut PARP1, respectively.
    Camptothecin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    LKT Laboratories camptothecin
    Detection of glutathionylated p53 protein in human tumor cells using anti p53-glut antibodies Induction of glut-p53 protein was determined in HCT116 cells after 0.4 mM H 2 O 2 treatment for different times (A), after 20 min incubation with H 2 O 2 (0.4 mM), TBH (0.6 mM) and diamide (0.6 mM) (B), with <t>camptothecin</t> (2 μM, 5h) (C) , and with camptothecin (2 μM), cisplatin (10 μM), and doxorubicin (5 μM) for 6 and 12 h (C). In the last four lanes of (A) and (B) and last lane of (C) the protein samples were treated with DTT before SDS-PAGE to reverse p53 glutathionylation. C, untreated control; CPT, camptothecin.
    Camptothecin, supplied by LKT Laboratories, used in various techniques. Bioz Stars score: 85/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abcam camptothecin
    Heatmap showing log 2 fold changes for treatments of staurosporine (STS), <t>camptothecin</t> <t>(CPT)</t> and copper oxide nanoparticles (CuO NPs) for the various time points ( n = 3), the levels of identification are indicated by 1 identified by reference substance (MS 2 and RT), 1a identified by reference substance (only RT), 2 identified by comparison of MS 2 spectra with reference databases such as Metlin. The dendrogram on the left hand side shows a clustering of similarly regulated metabolites upon treatment with STS/CPT/CuO NPs
    Camptothecin, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Insig2 inhibits apoptosis. ( a ) Three HCT116-Insig2-Myc clones and mock transfected HCT116 cells were cultured overnight, treated with 5 μM 5-FU and 50 ng/ml anti-Fas, or 1 μM camptothecin for 24 hr. Both floating and attached cells were

    Journal: International journal of cancer. Journal international du cancer

    Article Title: Insig2 is associated with colon tumorigenesis and inhibits Bax-mediated apoptosis

    doi: 10.1002/ijc.23510

    Figure Lengend Snippet: Insig2 inhibits apoptosis. ( a ) Three HCT116-Insig2-Myc clones and mock transfected HCT116 cells were cultured overnight, treated with 5 μM 5-FU and 50 ng/ml anti-Fas, or 1 μM camptothecin for 24 hr. Both floating and attached cells were

    Article Snippet: A variety of agents including etoposide (ETO, Sigma), 5-fluorouracil (5-FU, American Pharmaceutical Partners), camptothecin, staurosporine (STS) and Actinomycin D (Sigma) were used to induce apoptosis in HCT116 cells transfected with pcDNA3.1(+) (mock) or pCMV-Insig2-Myc.

    Techniques: Clone Assay, Transfection, Cell Culture

    Low levels of VRK2 sensitized cells to camptothecin treatment. ( a ) VRK2 knockdown was induced in A549 cells. These cells were treated with 5 μ M of camptothecin and the distribution of cytochrome c and Bax were determined in cytosolic and membrane fractions at different time points after camptothecin addition. Fractionated extracts were used for western blot analysis with the corresponding antibodies. ( b ) Low VRK2 facilitated activation of caspases in response to treatment with 5 μ M camptothecin. VRK2 knockdown was induced by two different siRNA, si-VRK2-06 (at the top) or si-VRK-2M (at the bottom), and caspase activation was determined by PARP processing. PARP was determined by immunoblot with a specific antibody at different time points after camptothecin addition to A549 cell culture. ( c ) Loss of VRK2 facilitated induction of DNA damage by camptothecin. A549 cells were transfected with si-Control or si-VRK2-06 and 96 hours later, they were treated with 5 μ M of camptothecin. A TUNEL assay was performed to detect the presence of free DNA ends at different time points. Cells labelled with fluorescein-12-dTUP (rTdT) and propidium iodide were analysed in a fluorescence microscope. Cell images with fluorescence are shown in the left panel and in the right panel the quantification of TUNEL positive cells are shown as a function of time following treatment with camptothecin. Si-Ct: si-control. siV2: si-VRK2-06

    Journal: Cell Death & Disease

    Article Title: Human VRK2 modulates apoptosis by interaction with Bcl-xL and regulation of BAX gene expression

    doi: 10.1038/cddis.2013.40

    Figure Lengend Snippet: Low levels of VRK2 sensitized cells to camptothecin treatment. ( a ) VRK2 knockdown was induced in A549 cells. These cells were treated with 5 μ M of camptothecin and the distribution of cytochrome c and Bax were determined in cytosolic and membrane fractions at different time points after camptothecin addition. Fractionated extracts were used for western blot analysis with the corresponding antibodies. ( b ) Low VRK2 facilitated activation of caspases in response to treatment with 5 μ M camptothecin. VRK2 knockdown was induced by two different siRNA, si-VRK2-06 (at the top) or si-VRK-2M (at the bottom), and caspase activation was determined by PARP processing. PARP was determined by immunoblot with a specific antibody at different time points after camptothecin addition to A549 cell culture. ( c ) Loss of VRK2 facilitated induction of DNA damage by camptothecin. A549 cells were transfected with si-Control or si-VRK2-06 and 96 hours later, they were treated with 5 μ M of camptothecin. A TUNEL assay was performed to detect the presence of free DNA ends at different time points. Cells labelled with fluorescein-12-dTUP (rTdT) and propidium iodide were analysed in a fluorescence microscope. Cell images with fluorescence are shown in the left panel and in the right panel the quantification of TUNEL positive cells are shown as a function of time following treatment with camptothecin. Si-Ct: si-control. siV2: si-VRK2-06

    Article Snippet: Chemicals Camptothecin and propidium iodide were from Sigma (St. Louis, MO, USA).

    Techniques: Western Blot, Activation Assay, Cell Culture, Transfection, TUNEL Assay, Fluorescence, Microscopy

    Effect of VRK2 knockdown on cell death induced by camptothecin treatment. VRK2 was knocked down in A549 cells and after 3 days, cells were treated with camptothecin for the indicated time points. ( a ) Effect of VRK2 knockdown on the sensitivity of A549 cells to camptothecin detected by alterations in the plasma membrane with annexin V+. Cells were stained with AnnexinV-FITC and positive cells were counted by flow cytometry. ( b ) Effect of VRK2 knockdown on A549 cell viability after camptothecin treatment. Viability was determined by Trypan blue dye exclusion assay. ( c ) Effect of VRK2 knockdown on mitochondrial membrane potential in the response to camptothecin was determined by loss of DIOC6 fluorescence in damaged cells. ( d ). Effect of camptothecin (5 μ M) on A549 cells and detection of propidium iodide staining by flow cytometry. The fragmented DNA was detected in the sub-G0/G1 window (bar). Si-Ct: si-control. siV2-06: si-VRK2-06

    Journal: Cell Death & Disease

    Article Title: Human VRK2 modulates apoptosis by interaction with Bcl-xL and regulation of BAX gene expression

    doi: 10.1038/cddis.2013.40

    Figure Lengend Snippet: Effect of VRK2 knockdown on cell death induced by camptothecin treatment. VRK2 was knocked down in A549 cells and after 3 days, cells were treated with camptothecin for the indicated time points. ( a ) Effect of VRK2 knockdown on the sensitivity of A549 cells to camptothecin detected by alterations in the plasma membrane with annexin V+. Cells were stained with AnnexinV-FITC and positive cells were counted by flow cytometry. ( b ) Effect of VRK2 knockdown on A549 cell viability after camptothecin treatment. Viability was determined by Trypan blue dye exclusion assay. ( c ) Effect of VRK2 knockdown on mitochondrial membrane potential in the response to camptothecin was determined by loss of DIOC6 fluorescence in damaged cells. ( d ). Effect of camptothecin (5 μ M) on A549 cells and detection of propidium iodide staining by flow cytometry. The fragmented DNA was detected in the sub-G0/G1 window (bar). Si-Ct: si-control. siV2-06: si-VRK2-06

    Article Snippet: Chemicals Camptothecin and propidium iodide were from Sigma (St. Louis, MO, USA).

    Techniques: Staining, Flow Cytometry, Cytometry, Exclusion Assay, Fluorescence

    DNA relaxation inhibition assay. Drug concentration was 10 µM. Activities are expressed relative to that of camptothecin (CPT): (+) 1–25%, (++) 26–50%, (+++) 51–75%, (++++) 76–100% of the CPT inhibition. All compounds were at least as potent as luotonin A and compound 3g showed a remarkable activity, comparable to that of camptothecin.

    Journal: PLoS ONE

    Article Title: B-Ring-Aryl Substituted Luotonin A Analogues with a New Binding Mode to the Topoisomerase 1-DNA Complex Show Enhanced Cytotoxic Activity

    doi: 10.1371/journal.pone.0095998

    Figure Lengend Snippet: DNA relaxation inhibition assay. Drug concentration was 10 µM. Activities are expressed relative to that of camptothecin (CPT): (+) 1–25%, (++) 26–50%, (+++) 51–75%, (++++) 76–100% of the CPT inhibition. All compounds were at least as potent as luotonin A and compound 3g showed a remarkable activity, comparable to that of camptothecin.

    Article Snippet: The bands were analysed using OriginPro 8.6 software (OriginLab, Northampton, Massachusetts) and the inhibitory activity of the drugs was calculated for each one as the supercoiled to unwinded bands ratio, and normalised to the activity of camptothecin (CPT) (Sigma-Aldrich, St. Louis, Missouri).

    Techniques: Inhibition, Concentration Assay, Cycling Probe Technology, Activity Assay

    Viability percentage (%) of B16F10 melanoma cells treated with camptothecin in the free control, conventional liposomes, and α-melanocyte-stimulating hormone (α-MSH) liposomes at a concentration of 50 μM. Each value represents the mean and standard deviation ( n = 3). * P

    Journal: Journal of Traditional and Complementary Medicine

    Article Title: Camptothecin-Loaded Liposomes with ?-Melanocyte-Stimulating Hormone Enhance Cytotoxicity Toward and Cellular Uptake by Melanomas: An Application of Nanomedicine on Natural Product

    doi: 10.4103/2225-4110.110423

    Figure Lengend Snippet: Viability percentage (%) of B16F10 melanoma cells treated with camptothecin in the free control, conventional liposomes, and α-melanocyte-stimulating hormone (α-MSH) liposomes at a concentration of 50 μM. Each value represents the mean and standard deviation ( n = 3). * P

    Article Snippet: Materials Camptothecin, cholesterol, stearylamine, α-MSH, dithiothreitol (DTT), rhodamine 123, and Sephadex G25 were purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Concentration Assay, Standard Deviation

    In vitro release percentage (%)–time profiles of camptothecin (at a 0.04% dose) across cellulose membranes from the free control (30% ethanol in double-distilled water), conventional liposomes, and α-melanocyte-stimulating hormone (α-MSH) liposomes. Each value represents the mean and standard deviation ( n = 4)

    Journal: Journal of Traditional and Complementary Medicine

    Article Title: Camptothecin-Loaded Liposomes with ?-Melanocyte-Stimulating Hormone Enhance Cytotoxicity Toward and Cellular Uptake by Melanomas: An Application of Nanomedicine on Natural Product

    doi: 10.4103/2225-4110.110423

    Figure Lengend Snippet: In vitro release percentage (%)–time profiles of camptothecin (at a 0.04% dose) across cellulose membranes from the free control (30% ethanol in double-distilled water), conventional liposomes, and α-melanocyte-stimulating hormone (α-MSH) liposomes. Each value represents the mean and standard deviation ( n = 4)

    Article Snippet: Materials Camptothecin, cholesterol, stearylamine, α-MSH, dithiothreitol (DTT), rhodamine 123, and Sephadex G25 were purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: In Vitro, Standard Deviation

    Viability percentage (%) of B16F10 melanoma cells treated with free camptothecin in DMSO at various concentrations. Each value represents the mean and standard deviation ( n = 3)

    Journal: Journal of Traditional and Complementary Medicine

    Article Title: Camptothecin-Loaded Liposomes with ?-Melanocyte-Stimulating Hormone Enhance Cytotoxicity Toward and Cellular Uptake by Melanomas: An Application of Nanomedicine on Natural Product

    doi: 10.4103/2225-4110.110423

    Figure Lengend Snippet: Viability percentage (%) of B16F10 melanoma cells treated with free camptothecin in DMSO at various concentrations. Each value represents the mean and standard deviation ( n = 3)

    Article Snippet: Materials Camptothecin, cholesterol, stearylamine, α-MSH, dithiothreitol (DTT), rhodamine 123, and Sephadex G25 were purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Standard Deviation

    FK866 affects NAD + (H) and ATP levels in Jurkat cells leading to cell death. a Flow-cytometric quantification of cell viability with AnnexinV (FITC) and 7AAD (PerCP-Cy5-5-A) staining. Jurkat cells were treated with FK866 for 48,72 and 120 h. Mock, 5 nM FK866 and 100 nM FK866 (at 48 and 72 h) are shown as representative samples. Flow-cytometry experiments were carried out on two biological replicates and statistics were based on the acquisition of 10000 events/sample. b Cell-cycle analysis with PI staining of the nuclei after 48 h of treatment. Overnight serum starvation is shown as positive control of induced cell cycle synchronization in G0/G1 phase. Histograms quantify the cell cycle phase distribution. Flow-cytometry experiments were carried out on two biological replicates and statistics were based on acquisition of 30000 events/sample. Cell cycle phase analysis was done using ModFit LT 3.2 software and the Sync Wizard model. c Jurkat cells were treated with FK866 for 48 h. Alternatively, cells were exposed to 5 μM of Camptothecin for 4 h as a positive control. Protease activity in cell extracts was assessed with a commercially available kit and values were normalized to the protein concentration in the same extracts. d Caspase 3/7 activity was measured in Jurkat cells treated as in c . e Jurkat cells were treated with FK866 for 48 h. Thereafter, intracellular NAD + (H) and ATP levels were evaluated in comparison with control Jurkat cells. RLUs were normalized to number of viable cells. In c - e the means with SD of at least three independent experiments are shown. Statistical significance was calculated with t -test (* and # indicates p -value

    Journal: BMC Cancer

    Article Title: EIF2A-dependent translational arrest protects leukemia cells from the energetic stress induced by NAMPT inhibition

    doi: 10.1186/s12885-015-1845-1

    Figure Lengend Snippet: FK866 affects NAD + (H) and ATP levels in Jurkat cells leading to cell death. a Flow-cytometric quantification of cell viability with AnnexinV (FITC) and 7AAD (PerCP-Cy5-5-A) staining. Jurkat cells were treated with FK866 for 48,72 and 120 h. Mock, 5 nM FK866 and 100 nM FK866 (at 48 and 72 h) are shown as representative samples. Flow-cytometry experiments were carried out on two biological replicates and statistics were based on the acquisition of 10000 events/sample. b Cell-cycle analysis with PI staining of the nuclei after 48 h of treatment. Overnight serum starvation is shown as positive control of induced cell cycle synchronization in G0/G1 phase. Histograms quantify the cell cycle phase distribution. Flow-cytometry experiments were carried out on two biological replicates and statistics were based on acquisition of 30000 events/sample. Cell cycle phase analysis was done using ModFit LT 3.2 software and the Sync Wizard model. c Jurkat cells were treated with FK866 for 48 h. Alternatively, cells were exposed to 5 μM of Camptothecin for 4 h as a positive control. Protease activity in cell extracts was assessed with a commercially available kit and values were normalized to the protein concentration in the same extracts. d Caspase 3/7 activity was measured in Jurkat cells treated as in c . e Jurkat cells were treated with FK866 for 48 h. Thereafter, intracellular NAD + (H) and ATP levels were evaluated in comparison with control Jurkat cells. RLUs were normalized to number of viable cells. In c - e the means with SD of at least three independent experiments are shown. Statistical significance was calculated with t -test (* and # indicates p -value

    Article Snippet: Chemicals FK866 (sc-205325) was bought from Santa Cruz, Compound C (P5499), Nicotinic acid (N0761), Actinomycin D (A9415), (S)-(+)-Camptothecin (C9911), Cycloheximide (C1988), MG-132 (M7449), Doxorubicin hydrochloride (D1515) and Dexamethasone (D4902) were bought from Sigma-Aldrich, CHS-828 (200484-11-3) from Cayman chemical, Torin 1 (S2827) and Rapamycin (S1039) from Selleck Chemicals, Cisplatin (ALX-400-040) from Enzo Life Sciences and Propidium Iodide Staining Solution from BD Pharmingen.

    Techniques: Flow Cytometry, Staining, Cytometry, Cell Cycle Assay, Positive Control, Software, Activity Assay, Protein Concentration

    Caspase-3 is activated in NPCs by camptothecin treatment and is blocked by GSK3 inhibitors a , NPCs in trophic factor-containing media were treated with 10 μ M camptothecin for 0, 2, 4, 6, or 8 h, and extracts were immunoblotted for p53, active cleaved caspase-3, cleaved PARP, and total GSK3 α/β as a loading control. b , p53 co-immunoprecipitated ( IP ) with GSK3 β after camptothecin ( CA ) treatment. C , control. c , p53, active caspase-3, cleaved PARP, and total GSK3 α / β were measured in NPCs 4 h after 10 μ M camptothecin treatment with no pretreatment ( Ctl ) or with a 30-min pretreatment with 20 mM lithium. d , active caspase-3, cleaved PARP, and β -actin were immunoblotted in NPCs 4 h after 10 μ M camptothecin treatment with no pretreatment ( Ctl ) or with a 30-min pretreatment with 20 mM lithium ( Li ), 5 μ M kenpaullone ( Kp ), 5 μ M GSK3 inhibitor II ( II ), or 20 μ M indirubin-3′-monoxime ( Ind ). e , quantitative values of the effects of GSK3 inhibitors on camptothecin-induced caspase-3 activation were obtained by densitometric measurements of immunoblots of active caspase-3 (means ± S.E.; n = 3; *, p

    Journal: The Journal of biological chemistry

    Article Title: Neural Precursor Cells Are Protected from Apoptosis Induced by Trophic Factor Withdrawal or Genotoxic Stress by Inhibitors of Glycogen Synthase Kinase 3 *Neural Precursor Cells Are Protected from Apoptosis Induced by Trophic Factor Withdrawal or Genotoxic Stress by Inhibitors of Glycogen Synthase Kinase 3 * , S

    doi: 10.1074/jbc.M702973200

    Figure Lengend Snippet: Caspase-3 is activated in NPCs by camptothecin treatment and is blocked by GSK3 inhibitors a , NPCs in trophic factor-containing media were treated with 10 μ M camptothecin for 0, 2, 4, 6, or 8 h, and extracts were immunoblotted for p53, active cleaved caspase-3, cleaved PARP, and total GSK3 α/β as a loading control. b , p53 co-immunoprecipitated ( IP ) with GSK3 β after camptothecin ( CA ) treatment. C , control. c , p53, active caspase-3, cleaved PARP, and total GSK3 α / β were measured in NPCs 4 h after 10 μ M camptothecin treatment with no pretreatment ( Ctl ) or with a 30-min pretreatment with 20 mM lithium. d , active caspase-3, cleaved PARP, and β -actin were immunoblotted in NPCs 4 h after 10 μ M camptothecin treatment with no pretreatment ( Ctl ) or with a 30-min pretreatment with 20 mM lithium ( Li ), 5 μ M kenpaullone ( Kp ), 5 μ M GSK3 inhibitor II ( II ), or 20 μ M indirubin-3′-monoxime ( Ind ). e , quantitative values of the effects of GSK3 inhibitors on camptothecin-induced caspase-3 activation were obtained by densitometric measurements of immunoblots of active caspase-3 (means ± S.E.; n = 3; *, p

    Article Snippet: DNA damage was induced by treating cells with 10 μ M camptothecin (Sigma) in NPC growth factor-containing media with FGF2.

    Techniques: Immunoprecipitation, CTL Assay, Activation Assay, Western Blot

    GSK3 inhibitors reduce camptothecin-induced apoptosis a , treatment with 20 mM lithium ( Li ), 5 μ M kenpaullone ( Kp ), 5 μ M GSK3 inhibitor II ( II ), or 20 μ M indirubin-3′-monoxime ( Ind ) attenuated camptothecin ( CA )-induced Bax activation (means ± S.E.; n = 3; *, p

    Journal: The Journal of biological chemistry

    Article Title: Neural Precursor Cells Are Protected from Apoptosis Induced by Trophic Factor Withdrawal or Genotoxic Stress by Inhibitors of Glycogen Synthase Kinase 3 *Neural Precursor Cells Are Protected from Apoptosis Induced by Trophic Factor Withdrawal or Genotoxic Stress by Inhibitors of Glycogen Synthase Kinase 3 * , S

    doi: 10.1074/jbc.M702973200

    Figure Lengend Snippet: GSK3 inhibitors reduce camptothecin-induced apoptosis a , treatment with 20 mM lithium ( Li ), 5 μ M kenpaullone ( Kp ), 5 μ M GSK3 inhibitor II ( II ), or 20 μ M indirubin-3′-monoxime ( Ind ) attenuated camptothecin ( CA )-induced Bax activation (means ± S.E.; n = 3; *, p

    Article Snippet: DNA damage was induced by treating cells with 10 μ M camptothecin (Sigma) in NPC growth factor-containing media with FGF2.

    Techniques: Activation Assay

    High-throughput assessment of fractional killing between conditions. (A,B) Assessment of live cell counts (mKate2 positive [mKate2 + ] objects) over lethal compound concentration-response series in two cell lines. Measurements were performed at 72 h. Btz: bortezomib, Vinb: vinblastine. Percent reduction in live cell counts between different compound concentrations is indicated by black lines and arrows and discussed in the main text. (C,D). Absolute metabolic viability measurements at select dose points. Colored circles in C and D correspond to specific doses indicated on the curves in A and B, respectively. (E,F). Metabolic viability measurements normalized per cell. Colored circles in E and F correspond to specific doses indicated on the curves in A and B, respectively. (G) Heatmap summaries of normalized, log 10 -scaled live mKate2 positive (mKate2+) live cell counts, SYTOX Green positive (SG+) dead cell counts, and integrated lethal fraction (Let. Frac.) scores over time (x-axis) and concentration (y-axis) for ten compounds in T98G N cells. For the mKate2 + heatmaps, live cell counts were normalized to 1 at time = 0 and expressed on a log scale. Values more than 10-fold lower than the starting values are colored black. Etop: etoposide, Cpt: camptothecin, Sts: staurosporine, Thap: thapsigargin, Tun: tunicamycin, Era: erastin, Pac, paclitaxel. (H) Live and dead cell counts for T98G N cells, extracted from the data shown in G. Data are from three independent experiments and represent mean ± SD (A-F, H) or mean (G).

    Journal: bioRxiv

    Article Title: Large-Scale Analysis of Cell Death Phenotypic Heterogeneity

    doi: 10.1101/2020.02.28.970079

    Figure Lengend Snippet: High-throughput assessment of fractional killing between conditions. (A,B) Assessment of live cell counts (mKate2 positive [mKate2 + ] objects) over lethal compound concentration-response series in two cell lines. Measurements were performed at 72 h. Btz: bortezomib, Vinb: vinblastine. Percent reduction in live cell counts between different compound concentrations is indicated by black lines and arrows and discussed in the main text. (C,D). Absolute metabolic viability measurements at select dose points. Colored circles in C and D correspond to specific doses indicated on the curves in A and B, respectively. (E,F). Metabolic viability measurements normalized per cell. Colored circles in E and F correspond to specific doses indicated on the curves in A and B, respectively. (G) Heatmap summaries of normalized, log 10 -scaled live mKate2 positive (mKate2+) live cell counts, SYTOX Green positive (SG+) dead cell counts, and integrated lethal fraction (Let. Frac.) scores over time (x-axis) and concentration (y-axis) for ten compounds in T98G N cells. For the mKate2 + heatmaps, live cell counts were normalized to 1 at time = 0 and expressed on a log scale. Values more than 10-fold lower than the starting values are colored black. Etop: etoposide, Cpt: camptothecin, Sts: staurosporine, Thap: thapsigargin, Tun: tunicamycin, Era: erastin, Pac, paclitaxel. (H) Live and dead cell counts for T98G N cells, extracted from the data shown in G. Data are from three independent experiments and represent mean ± SD (A-F, H) or mean (G).

    Article Snippet: Chemicals and reagents Camptothecin (Cat# S1288), vinblastine (Cat# S1248), pimasertib (Cat# S1457), trametinib (Cat# S2673), ABT-737 (Cat# S1002), A-1155463 (Cat# S7800), Nutlin-3 (Cat# S1061) were purchased from Selleck Chemicals (Houston, TX).

    Techniques: High Throughput Screening Assay, Concentration Assay

    Quantification of time-dependent fractional killing. (A) Overview of cell death kinetic analysis using time-lapse imaging and curve fitting. Lethal fraction curves can be fit with a lag exponential death (LED) model that yields two key parameter values, D O and D R , which reflect the timing of cell death onset and the maximum rate of cell death within the population, respectively ( Forcina et al., 2017 ). (B) Treatments that trigger cell death with low between-cell heterogeneity will have high D R values, and vice versa, which can impact the interpretation of FK at any given early timepoint. (C) Extracted D R parameter values for LED curves fit to lethal fraction scores for a sub-set of the lethal compounds shown in Figure 1D and S1G . Kinetic parameter values were only computed for compound concentrations where the lethal fraction at 120 h exceeded 0.5. Results are mean ± SD from three independent experiments. (D) Representative population images over time for camptothecin (Cpt)-treated cells. Heterogeneity is especially apparent at the 48 h timepoint, where arrows indicate example live cells and arrowheads indicate example dead cells. Scale bar = 75 µm. (E) Traces of lethal fraction scores over time for 140 lethal compounds identified previously. (F) Extracted D R values for 139 lethal compounds from E where it was possible to compute this parameter value. (G) D R values for compounds in F broken down by compound class. (H) Representative population images for select conditions from G to illustrate heterogeneity in cell death initiation throughout the population. Scale bar = 75 µm.

    Journal: bioRxiv

    Article Title: Large-Scale Analysis of Cell Death Phenotypic Heterogeneity

    doi: 10.1101/2020.02.28.970079

    Figure Lengend Snippet: Quantification of time-dependent fractional killing. (A) Overview of cell death kinetic analysis using time-lapse imaging and curve fitting. Lethal fraction curves can be fit with a lag exponential death (LED) model that yields two key parameter values, D O and D R , which reflect the timing of cell death onset and the maximum rate of cell death within the population, respectively ( Forcina et al., 2017 ). (B) Treatments that trigger cell death with low between-cell heterogeneity will have high D R values, and vice versa, which can impact the interpretation of FK at any given early timepoint. (C) Extracted D R parameter values for LED curves fit to lethal fraction scores for a sub-set of the lethal compounds shown in Figure 1D and S1G . Kinetic parameter values were only computed for compound concentrations where the lethal fraction at 120 h exceeded 0.5. Results are mean ± SD from three independent experiments. (D) Representative population images over time for camptothecin (Cpt)-treated cells. Heterogeneity is especially apparent at the 48 h timepoint, where arrows indicate example live cells and arrowheads indicate example dead cells. Scale bar = 75 µm. (E) Traces of lethal fraction scores over time for 140 lethal compounds identified previously. (F) Extracted D R values for 139 lethal compounds from E where it was possible to compute this parameter value. (G) D R values for compounds in F broken down by compound class. (H) Representative population images for select conditions from G to illustrate heterogeneity in cell death initiation throughout the population. Scale bar = 75 µm.

    Article Snippet: Chemicals and reagents Camptothecin (Cat# S1288), vinblastine (Cat# S1248), pimasertib (Cat# S1457), trametinib (Cat# S2673), ABT-737 (Cat# S1002), A-1155463 (Cat# S7800), Nutlin-3 (Cat# S1061) were purchased from Selleck Chemicals (Houston, TX).

    Techniques: Imaging

    Systematic investigation of fractional killing. (A) At a given drug dose and time, a fraction of cells within the population will be killed while others survive. (B) Overview of cell death analysis using time lapse imaging of population live (nuclear mKate2) and dead (SYTOX Green) cell over time. These data can be integrated into a single metric, the lethal fraction, representing overall population cell death. (C) Heatmap summaries of mKate2 positive (mKate2 + ) live cell counts, SYTOX Green positive (SG + ) dead cell counts, and integrated lethal fraction scores as a function of time (x-axis) and concentration (y-axis) for ten compounds in U-2 OS N cells. For the mKate2 + heatmaps, live cell counts were normalized to 1 at time = 0 and expressed on a log 10 scale. Etop: etoposide, Cpt: camptothecin, Sts: staurosporine, Thap: thapsigargin, Tun: tunicamycin, Era: erastin, Pac, paclitaxel, Vinb: vinblastine. (D) Isolated live cell counts, dead cell counts (mKate2 + or SG + objects/mm 2 imaged area; Obj./mm 2 ) and corresponding lethal fraction (Let. frac.) scores over time, extracted from select conditions in C. The stars (*) indicate where live cell counts increase beyond the range of the y-axis scale. (E, F). Lethal fraction scores over time for U-2 OS N (E) and T98G N (F) cells exposed to four lethal compounds. (G, H). Extracted mean lethal fractions at select timepoints from the data presented in E and F. Data are from three independent experiments and represent the mean (C, G, H) or mean ± SD (D-F).

    Journal: bioRxiv

    Article Title: Large-Scale Analysis of Cell Death Phenotypic Heterogeneity

    doi: 10.1101/2020.02.28.970079

    Figure Lengend Snippet: Systematic investigation of fractional killing. (A) At a given drug dose and time, a fraction of cells within the population will be killed while others survive. (B) Overview of cell death analysis using time lapse imaging of population live (nuclear mKate2) and dead (SYTOX Green) cell over time. These data can be integrated into a single metric, the lethal fraction, representing overall population cell death. (C) Heatmap summaries of mKate2 positive (mKate2 + ) live cell counts, SYTOX Green positive (SG + ) dead cell counts, and integrated lethal fraction scores as a function of time (x-axis) and concentration (y-axis) for ten compounds in U-2 OS N cells. For the mKate2 + heatmaps, live cell counts were normalized to 1 at time = 0 and expressed on a log 10 scale. Etop: etoposide, Cpt: camptothecin, Sts: staurosporine, Thap: thapsigargin, Tun: tunicamycin, Era: erastin, Pac, paclitaxel, Vinb: vinblastine. (D) Isolated live cell counts, dead cell counts (mKate2 + or SG + objects/mm 2 imaged area; Obj./mm 2 ) and corresponding lethal fraction (Let. frac.) scores over time, extracted from select conditions in C. The stars (*) indicate where live cell counts increase beyond the range of the y-axis scale. (E, F). Lethal fraction scores over time for U-2 OS N (E) and T98G N (F) cells exposed to four lethal compounds. (G, H). Extracted mean lethal fractions at select timepoints from the data presented in E and F. Data are from three independent experiments and represent the mean (C, G, H) or mean ± SD (D-F).

    Article Snippet: Chemicals and reagents Camptothecin (Cat# S1288), vinblastine (Cat# S1248), pimasertib (Cat# S1457), trametinib (Cat# S2673), ABT-737 (Cat# S1002), A-1155463 (Cat# S7800), Nutlin-3 (Cat# S1061) were purchased from Selleck Chemicals (Houston, TX).

    Techniques: Imaging, Concentration Assay, Isolation

    (A) Cell death in response to treatment with pimasertib (Pim, 5 µM), trametinib (Tram, 5 µM), bortezomib (Btz, 50 nM), camptothecin (Cpt, 1 µM) ± cycloheximide (CHX). (B) Cell death ± Q-VD-OPh (50 µM). Pim, Tram and Btz treatments are as in A. (C) Protein expression ± pimasertib (5 µM, 48 h). (D) Expression of BIM in response to doxycycline (Dox). (E) Cell death over time ± Pim combined with Dox-inducible expression of wild-type BIM. (F) Cell death kinetic parameters computed from the data in E. (G) Expression of the inactive BIM G156E mutant + Dox. (H) Cell death over time in response to Dox-inducible expression of mutant BIM G156E + Pim. (I) Cell death in Calu-6 N cells in response to compound treatment ± the selective MCL1 inhibitor S63845 (5 µM). (J) Cell death kinetic parameters computed from the data in I. (K) Expression of MCL1 in A549 N;Cas9 cells transduced with short guide RNAs against GFP or MCL1 . (L) Cell death over time in the cells lines from K, treated as indicated. D R values computed from each curve are indicated for MEKis. Data are from at least three independent experiments and represent mean ± SD (A,B,E,H,I,L) or mean ± 95% C.I (F,J).

    Journal: bioRxiv

    Article Title: Large-Scale Analysis of Cell Death Phenotypic Heterogeneity

    doi: 10.1101/2020.02.28.970079

    Figure Lengend Snippet: (A) Cell death in response to treatment with pimasertib (Pim, 5 µM), trametinib (Tram, 5 µM), bortezomib (Btz, 50 nM), camptothecin (Cpt, 1 µM) ± cycloheximide (CHX). (B) Cell death ± Q-VD-OPh (50 µM). Pim, Tram and Btz treatments are as in A. (C) Protein expression ± pimasertib (5 µM, 48 h). (D) Expression of BIM in response to doxycycline (Dox). (E) Cell death over time ± Pim combined with Dox-inducible expression of wild-type BIM. (F) Cell death kinetic parameters computed from the data in E. (G) Expression of the inactive BIM G156E mutant + Dox. (H) Cell death over time in response to Dox-inducible expression of mutant BIM G156E + Pim. (I) Cell death in Calu-6 N cells in response to compound treatment ± the selective MCL1 inhibitor S63845 (5 µM). (J) Cell death kinetic parameters computed from the data in I. (K) Expression of MCL1 in A549 N;Cas9 cells transduced with short guide RNAs against GFP or MCL1 . (L) Cell death over time in the cells lines from K, treated as indicated. D R values computed from each curve are indicated for MEKis. Data are from at least three independent experiments and represent mean ± SD (A,B,E,H,I,L) or mean ± 95% C.I (F,J).

    Article Snippet: Chemicals and reagents Camptothecin (Cat# S1288), vinblastine (Cat# S1248), pimasertib (Cat# S1457), trametinib (Cat# S2673), ABT-737 (Cat# S1002), A-1155463 (Cat# S7800), Nutlin-3 (Cat# S1061) were purchased from Selleck Chemicals (Houston, TX).

    Techniques: Expressing, Mutagenesis, Transduction

    Investigating the MEKi-induced fractional killing. (A) Cell death summarized over time and MEKi concentration. Data are mean values from three independent experiments. Cell death kinetic parameters for select concentrations indicated by the symbols are shown at right, with 95% confidence intervals in brackets. nd: not determined. (B) Cell death summarized over time and SCH772984 concentration. Data are mean values from three independent experiments. Cell death kinetic parameters for one concentration indicated by the black circle are shown, with 95% confidence intervals in brackets. nd: not determined. (C) Phospho-ERK1/2 (pERK), p53 and p21 levels in A549 N cells ± nutlin-3 (10 µM, 48 h). (D) Cell death over time following pretreatment (2 day) ± nutlin-3 (10 µM). +Cmpd indicates the time of pimasertib (Pim, 5 µM), trametinib (Tram, 5 µM) or camptothecin (Cpt, 10 µM) addition. p53 stabilization has no effect on the kinetics of MEKi-induced cell death. (E) Analysis of cell cycle phase by PI/FACS ± nutlin-3 (10 µM) for the indicated times. (F) Cell death ± pimasertib ± vehicle (ethanol, EtOH), 2-deoxyglucose (2-DG, 10 mM) or oligomycin (Olig, 100 nM). Disruption of bioenergetics has no effect on FK. (G) Oxygen consumption (OCR) and extracellular acidification (ECAR) determined using a Seahorse assay in A549 N cells ± 2-DG or Olig as in F. (H) Outline of FACS-based scheme to isolate cells with high or low intracellular ROS as assessed using H 2 DCFDA. (I) Cell death over time for isolated H 2 DCFDA High and H 2 DCFDA Low sub-populations exposed to pimasertib (5 µM). (J) Cell death over time in A549 N cells ± MEKis ± N-acetylcysteine (NAC, 1 mM). Erastin2 is a positive control compound that induces oxidative cell death. Data in D-G, I and J are mean ± SD from at least three independent experiments.

    Journal: bioRxiv

    Article Title: Large-Scale Analysis of Cell Death Phenotypic Heterogeneity

    doi: 10.1101/2020.02.28.970079

    Figure Lengend Snippet: Investigating the MEKi-induced fractional killing. (A) Cell death summarized over time and MEKi concentration. Data are mean values from three independent experiments. Cell death kinetic parameters for select concentrations indicated by the symbols are shown at right, with 95% confidence intervals in brackets. nd: not determined. (B) Cell death summarized over time and SCH772984 concentration. Data are mean values from three independent experiments. Cell death kinetic parameters for one concentration indicated by the black circle are shown, with 95% confidence intervals in brackets. nd: not determined. (C) Phospho-ERK1/2 (pERK), p53 and p21 levels in A549 N cells ± nutlin-3 (10 µM, 48 h). (D) Cell death over time following pretreatment (2 day) ± nutlin-3 (10 µM). +Cmpd indicates the time of pimasertib (Pim, 5 µM), trametinib (Tram, 5 µM) or camptothecin (Cpt, 10 µM) addition. p53 stabilization has no effect on the kinetics of MEKi-induced cell death. (E) Analysis of cell cycle phase by PI/FACS ± nutlin-3 (10 µM) for the indicated times. (F) Cell death ± pimasertib ± vehicle (ethanol, EtOH), 2-deoxyglucose (2-DG, 10 mM) or oligomycin (Olig, 100 nM). Disruption of bioenergetics has no effect on FK. (G) Oxygen consumption (OCR) and extracellular acidification (ECAR) determined using a Seahorse assay in A549 N cells ± 2-DG or Olig as in F. (H) Outline of FACS-based scheme to isolate cells with high or low intracellular ROS as assessed using H 2 DCFDA. (I) Cell death over time for isolated H 2 DCFDA High and H 2 DCFDA Low sub-populations exposed to pimasertib (5 µM). (J) Cell death over time in A549 N cells ± MEKis ± N-acetylcysteine (NAC, 1 mM). Erastin2 is a positive control compound that induces oxidative cell death. Data in D-G, I and J are mean ± SD from at least three independent experiments.

    Article Snippet: Chemicals and reagents Camptothecin (Cat# S1288), vinblastine (Cat# S1248), pimasertib (Cat# S1457), trametinib (Cat# S2673), ABT-737 (Cat# S1002), A-1155463 (Cat# S7800), Nutlin-3 (Cat# S1061) were purchased from Selleck Chemicals (Houston, TX).

    Techniques: Concentration Assay, FACS, Isolation, Positive Control

    A. Apoptotic and B. proliferative behavior of VHL wt and mutated RCC4 cells after treatment with Camptothecin, Camptothecin and Sunitinib (Combination), and Sunitinib. Signals after treatment of each cell line were normalized with corresponding mock signals (without treatment). VHL 30 was used as reference and compared to the other cell lines. p-value: *

    Journal: Oncotarget

    Article Title: VHL missense mutations in the p53 binding domain show different effects on p53 signaling and HIFα degradation in clear cell renal cell carcinoma

    doi: 10.18632/oncotarget.14372

    Figure Lengend Snippet: A. Apoptotic and B. proliferative behavior of VHL wt and mutated RCC4 cells after treatment with Camptothecin, Camptothecin and Sunitinib (Combination), and Sunitinib. Signals after treatment of each cell line were normalized with corresponding mock signals (without treatment). VHL 30 was used as reference and compared to the other cell lines. p-value: *

    Article Snippet: Drug treatment Camptothecin and Sunitinib (Selleck Chemicals, USA) were diluted in DMSO at 0.05 μM and 10 μM concentration, respectively, and applied to the cells alone or in combination for 48h at a final concentration of 0.3% DMSO following the manufacturer recommendation.

    Techniques:

    Effects of A. argyi extract on the function of T lymphocytes. For (A , B) , lymphocytes (2 × 10 5 ) were left unstimulated (unstim.) or were activated with anti-CD3 and anti-CD28 mAbs (100 ng each and incubated for 24 h with medium (unstim., stim.), cyclosporine A (CsA; 4.16 µM), camptothecin (CPT; 300 µM), or the A. argyi extract. Cells were stained with anti-CD69-FITC and anti-CD4-APC (A) or anti-CD25-PE and anti-CD4-APC (B) . Expression of surface markers (CD69, CD25, CD4) was analyzed by flow cytometry, and the amounts of activated CD4 + and CD4 – T lymphocytes were determined. For (C , D) expanded lymphocytes (2 × 10 5 ) were incubated for 20 h with medium (unstim., stim.), cyclosporine A (CsA; 4.16 µM), camptothecin (CPT; 300 µM), parthenolide (20 µM) or the A. argyi extract. Subsequently, the cells were stimulated with PMA/Ionomycin for 4 h at 37°C. IL-2 (C) and IFN-γ (D) were quantified in the supernatants by ELISA. The results of three different experiments are summarized and are depicted as mean ± SD in relation to the untreated, stimulated control. *p

    Journal: Frontiers in Pharmacology

    Article Title: Immunosuppressive Activity of Artemisia argyi Extract and Isolated Compounds

    doi: 10.3389/fphar.2020.00402

    Figure Lengend Snippet: Effects of A. argyi extract on the function of T lymphocytes. For (A , B) , lymphocytes (2 × 10 5 ) were left unstimulated (unstim.) or were activated with anti-CD3 and anti-CD28 mAbs (100 ng each and incubated for 24 h with medium (unstim., stim.), cyclosporine A (CsA; 4.16 µM), camptothecin (CPT; 300 µM), or the A. argyi extract. Cells were stained with anti-CD69-FITC and anti-CD4-APC (A) or anti-CD25-PE and anti-CD4-APC (B) . Expression of surface markers (CD69, CD25, CD4) was analyzed by flow cytometry, and the amounts of activated CD4 + and CD4 – T lymphocytes were determined. For (C , D) expanded lymphocytes (2 × 10 5 ) were incubated for 20 h with medium (unstim., stim.), cyclosporine A (CsA; 4.16 µM), camptothecin (CPT; 300 µM), parthenolide (20 µM) or the A. argyi extract. Subsequently, the cells were stimulated with PMA/Ionomycin for 4 h at 37°C. IL-2 (C) and IFN-γ (D) were quantified in the supernatants by ELISA. The results of three different experiments are summarized and are depicted as mean ± SD in relation to the untreated, stimulated control. *p

    Article Snippet: Activation and Treatment of Lymphocytes Lymphocytes were activated with anti-CD3 (clone OKT3) and anti-CD28 (clone 28.2) mAbs (each 100 ng/ml; both from eBioscience, Frankfurt, Germany) in the presence of medium; cyclosporine A (CsA; 4.16 µM; Sandimmun 50 mg/ml, Novartis Pharma, Basel, Switzerland); camptothecin (CPT; 300 µM: Tocris, Bristol, UK); 0.5% Triton-X 100; plant extract; or isolated compounds from A. argyi , as described previously ( ).

    Techniques: Incubation, Staining, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay

    Overview of recently published results (A) and effect of the A. argyi extract on the viability of T lymphocytes (B, C) . Expanded human lymphocytes (2 × 10 5 ) were left unstimulated (unstim.) or were stimulated (stim.) with anti-CD3 and anti-CD28 mAbs (100 ng each) and incubated for 48 h with medium (unstim., stim.), camptothecin (CPT; 300 μM), Triton-X 100 (0.5%) or the A. argyi extract. Annexin V-FITC and PI double stainings were performed after incubation. The proportions of viable, necrotic, and apoptotic cells were determined by flow cytometry. Data are depicted as dot plots (B) , and the proportion of apoptotic/necrotic lymphocytes in relation to the stimulated control was determined from two independent experiments. Data are depicted as mean ± SD (C) .

    Journal: Frontiers in Pharmacology

    Article Title: Immunosuppressive Activity of Artemisia argyi Extract and Isolated Compounds

    doi: 10.3389/fphar.2020.00402

    Figure Lengend Snippet: Overview of recently published results (A) and effect of the A. argyi extract on the viability of T lymphocytes (B, C) . Expanded human lymphocytes (2 × 10 5 ) were left unstimulated (unstim.) or were stimulated (stim.) with anti-CD3 and anti-CD28 mAbs (100 ng each) and incubated for 48 h with medium (unstim., stim.), camptothecin (CPT; 300 μM), Triton-X 100 (0.5%) or the A. argyi extract. Annexin V-FITC and PI double stainings were performed after incubation. The proportions of viable, necrotic, and apoptotic cells were determined by flow cytometry. Data are depicted as dot plots (B) , and the proportion of apoptotic/necrotic lymphocytes in relation to the stimulated control was determined from two independent experiments. Data are depicted as mean ± SD (C) .

    Article Snippet: Activation and Treatment of Lymphocytes Lymphocytes were activated with anti-CD3 (clone OKT3) and anti-CD28 (clone 28.2) mAbs (each 100 ng/ml; both from eBioscience, Frankfurt, Germany) in the presence of medium; cyclosporine A (CsA; 4.16 µM; Sandimmun 50 mg/ml, Novartis Pharma, Basel, Switzerland); camptothecin (CPT; 300 µM: Tocris, Bristol, UK); 0.5% Triton-X 100; plant extract; or isolated compounds from A. argyi , as described previously ( ).

    Techniques: Incubation, Flow Cytometry

    Effects of kalata B1 on cytotoxicity of activated human peripheral blood mononuclear cells. Human primary PBMC were activated with anti-CD3/CD28 mAbs in the presence of medium (ctrl), camptothecin (CPT, 30 μ g/mL), Triton-X 100 (T-x), or different concentrations of kalata B1 (1.8–14 μ M) and analyzed for “subG1” DNA content (A) by flow cytometry. Cells were stained with annexin V and propidium iodide (PI) to assess the percentages of viable (annexin V − /PI − ), apoptotic (annexin V + /PI − or annexin V + /PI + ), and necrotic (annexin V − /PI + ) cells. Dot plots were analyzed, and representative graphs are shown (B). Results from three independent experiments are summarized, and data are presented as mean ± SEM (C and D). n.d. = not detectable.

    Journal: Journal of natural products

    Article Title: Do Plant Cyclotides Have Potential As Immunosuppressant Peptides?

    doi: 10.1021/np200722w

    Figure Lengend Snippet: Effects of kalata B1 on cytotoxicity of activated human peripheral blood mononuclear cells. Human primary PBMC were activated with anti-CD3/CD28 mAbs in the presence of medium (ctrl), camptothecin (CPT, 30 μ g/mL), Triton-X 100 (T-x), or different concentrations of kalata B1 (1.8–14 μ M) and analyzed for “subG1” DNA content (A) by flow cytometry. Cells were stained with annexin V and propidium iodide (PI) to assess the percentages of viable (annexin V − /PI − ), apoptotic (annexin V + /PI − or annexin V + /PI + ), and necrotic (annexin V − /PI + ) cells. Dot plots were analyzed, and representative graphs are shown (B). Results from three independent experiments are summarized, and data are presented as mean ± SEM (C and D). n.d. = not detectable.

    Article Snippet: PBMC (105 ) were stimulated with anti-human CD3 (clone OKT3) and anti-human CD28 (clone 28.2) mAbs (both from eBioscience, Frankfurt, Germany) for 72 h in the presence of medium, the control agents camptothecin (CPT; 30 μ g/mL: Tocris, Eching, Germany), and Triton-X 100 (0.5%; Carl Roth, Karlsruhe, Germany) or different concentrations of O. affinis extract, melittin (PolyPeptide, Strasbourg, France), or kalata B1, respectively.

    Techniques: Cycling Probe Technology, Flow Cytometry, Cytometry, Staining

    Effects of the O. affinis cyclotide extract on cell proliferation of activated human peripheral blood mononuclear cells. CFSE-labeled primary human PBMC were antibody-activated (anti-CD3/CD28 mAbs) and cultured in the presence of medium (ctrl), camptothecin (CPT, 30 μ g/mL), or different concentrations (50–100 μ g/mL) of O. affinis cyclotide extract. The cells were further analyzed for cell viability and proliferation capacity using flow forward-side-scatter-based flow cytometric analysis (A and B). Cell division analyses were assessed by FACS and illustrated as representative dot plots (C). Results are summarized from three independent experiments in (D), and data are presented as mean ± SEM.

    Journal: Journal of natural products

    Article Title: Do Plant Cyclotides Have Potential As Immunosuppressant Peptides?

    doi: 10.1021/np200722w

    Figure Lengend Snippet: Effects of the O. affinis cyclotide extract on cell proliferation of activated human peripheral blood mononuclear cells. CFSE-labeled primary human PBMC were antibody-activated (anti-CD3/CD28 mAbs) and cultured in the presence of medium (ctrl), camptothecin (CPT, 30 μ g/mL), or different concentrations (50–100 μ g/mL) of O. affinis cyclotide extract. The cells were further analyzed for cell viability and proliferation capacity using flow forward-side-scatter-based flow cytometric analysis (A and B). Cell division analyses were assessed by FACS and illustrated as representative dot plots (C). Results are summarized from three independent experiments in (D), and data are presented as mean ± SEM.

    Article Snippet: PBMC (105 ) were stimulated with anti-human CD3 (clone OKT3) and anti-human CD28 (clone 28.2) mAbs (both from eBioscience, Frankfurt, Germany) for 72 h in the presence of medium, the control agents camptothecin (CPT; 30 μ g/mL: Tocris, Eching, Germany), and Triton-X 100 (0.5%; Carl Roth, Karlsruhe, Germany) or different concentrations of O. affinis extract, melittin (PolyPeptide, Strasbourg, France), or kalata B1, respectively.

    Techniques: Labeling, Cell Culture, Cycling Probe Technology, Flow Cytometry, FACS

    Effects of kalata B1 on cell proliferation of activated human peripheral blood mononuclear cells. The influence of medium (ctrl), camptothecin (CPT, 30 μ g/mL), or different concentrations of kalata B1 (1.8–14 μ M) on proliferation of CFSE + anti-CD3/CD28 mAbs-activated human primary PBMC was measured by cell division analysis using flow cytometry. Data are presented as mean ± SEM of four independent experiments.

    Journal: Journal of natural products

    Article Title: Do Plant Cyclotides Have Potential As Immunosuppressant Peptides?

    doi: 10.1021/np200722w

    Figure Lengend Snippet: Effects of kalata B1 on cell proliferation of activated human peripheral blood mononuclear cells. The influence of medium (ctrl), camptothecin (CPT, 30 μ g/mL), or different concentrations of kalata B1 (1.8–14 μ M) on proliferation of CFSE + anti-CD3/CD28 mAbs-activated human primary PBMC was measured by cell division analysis using flow cytometry. Data are presented as mean ± SEM of four independent experiments.

    Article Snippet: PBMC (105 ) were stimulated with anti-human CD3 (clone OKT3) and anti-human CD28 (clone 28.2) mAbs (both from eBioscience, Frankfurt, Germany) for 72 h in the presence of medium, the control agents camptothecin (CPT; 30 μ g/mL: Tocris, Eching, Germany), and Triton-X 100 (0.5%; Carl Roth, Karlsruhe, Germany) or different concentrations of O. affinis extract, melittin (PolyPeptide, Strasbourg, France), or kalata B1, respectively.

    Techniques: Cycling Probe Technology, Flow Cytometry, Cytometry

    Effects of siRNAs specific to CXXC5 on cellular growth. MCF7 cells grown in 10% CD-FBS containing medium for 48 h were transiently transfected without (UT) or with 10 nM CtS or siRNA#10 in the absence (0.01% ethanol) or the presence of 10 −8 M E2 up to 72 h. ( a ) Cells were subjected to cell counting using a hemocytometer. Results, as the mean ± S.E. of three independent determinations, depict fold change in cellular growth compared with those observed with cells at time 0 in the absence of E2, which is set to 1. In ( a–f ) the asterisk with a superscript “a” indicates significant difference from the corresponding ethanol-treated group (-E2); whereas superscript “b” depicts a significant difference from CtS transfected cells in the presence of E2. ( b ) The cDNA library generated from total RNA (50 ng) isolated from transfected cells for 48 h was subjected to qPCR using a primer set specific to CXXC5. ( c ) Nuclear extracts from untransfected (UT) or transfected cells were subjected to WB using an antibody for CXXC5, ERα or HDAC1. NS denotes non-specific protein; Molecular mass in KDa is indicated. Transfected cells were subjected to cell counting ( d ) and MTT assay ( e ). In ( b , d , e) , results depicting the mean ± S.E. of three independent determinations indicate fold change in mRNA levels ( b ) or cell numbers ( d , e ) compared with CtS in the absence of E2, which is set to 1. Transfected MCF7 cells were subjected to flow cytometry ( f ) with results depicting the percent of cells in the G1, G2 and S phases, or to Annexin V assay followed by flow cytometry ( g ). In ( f , g ), results are the mean ± SEM of three independent experiments. ( h i ) Untransfected (UT) or transfected cells were also subjected to the cell death inducer camptothecin (2 nM) for 24 h without (−E2) or with E2 (E2). Cells were then subjected to Annexin V assay ( h ) with results as the mean ± S.E. of three independent experiments depicting percent change in apoptotic (upper right quadrant) and death (upper left quadrant) cells, or to WB ( i ) using a PARP1-specific antibody. P and CP denote the uncut and cut PARP1, respectively.

    Journal: Scientific Reports

    Article Title: CXXC5 as an unmethylated CpG dinucleotide binding protein contributes to estrogen-mediated cellular proliferation

    doi: 10.1038/s41598-020-62912-0

    Figure Lengend Snippet: Effects of siRNAs specific to CXXC5 on cellular growth. MCF7 cells grown in 10% CD-FBS containing medium for 48 h were transiently transfected without (UT) or with 10 nM CtS or siRNA#10 in the absence (0.01% ethanol) or the presence of 10 −8 M E2 up to 72 h. ( a ) Cells were subjected to cell counting using a hemocytometer. Results, as the mean ± S.E. of three independent determinations, depict fold change in cellular growth compared with those observed with cells at time 0 in the absence of E2, which is set to 1. In ( a–f ) the asterisk with a superscript “a” indicates significant difference from the corresponding ethanol-treated group (-E2); whereas superscript “b” depicts a significant difference from CtS transfected cells in the presence of E2. ( b ) The cDNA library generated from total RNA (50 ng) isolated from transfected cells for 48 h was subjected to qPCR using a primer set specific to CXXC5. ( c ) Nuclear extracts from untransfected (UT) or transfected cells were subjected to WB using an antibody for CXXC5, ERα or HDAC1. NS denotes non-specific protein; Molecular mass in KDa is indicated. Transfected cells were subjected to cell counting ( d ) and MTT assay ( e ). In ( b , d , e) , results depicting the mean ± S.E. of three independent determinations indicate fold change in mRNA levels ( b ) or cell numbers ( d , e ) compared with CtS in the absence of E2, which is set to 1. Transfected MCF7 cells were subjected to flow cytometry ( f ) with results depicting the percent of cells in the G1, G2 and S phases, or to Annexin V assay followed by flow cytometry ( g ). In ( f , g ), results are the mean ± SEM of three independent experiments. ( h i ) Untransfected (UT) or transfected cells were also subjected to the cell death inducer camptothecin (2 nM) for 24 h without (−E2) or with E2 (E2). Cells were then subjected to Annexin V assay ( h ) with results as the mean ± S.E. of three independent experiments depicting percent change in apoptotic (upper right quadrant) and death (upper left quadrant) cells, or to WB ( i ) using a PARP1-specific antibody. P and CP denote the uncut and cut PARP1, respectively.

    Article Snippet: Camptothecin (13637) was purchased from Cell Signaling Technology.

    Techniques: Transfection, Cell Counting, cDNA Library Assay, Generated, Isolation, Real-time Polymerase Chain Reaction, Western Blot, MTT Assay, Flow Cytometry, Annexin V Assay

    Detection of glutathionylated p53 protein in human tumor cells using anti p53-glut antibodies Induction of glut-p53 protein was determined in HCT116 cells after 0.4 mM H 2 O 2 treatment for different times (A), after 20 min incubation with H 2 O 2 (0.4 mM), TBH (0.6 mM) and diamide (0.6 mM) (B), with camptothecin (2 μM, 5h) (C) , and with camptothecin (2 μM), cisplatin (10 μM), and doxorubicin (5 μM) for 6 and 12 h (C). In the last four lanes of (A) and (B) and last lane of (C) the protein samples were treated with DTT before SDS-PAGE to reverse p53 glutathionylation. C, untreated control; CPT, camptothecin.

    Journal: Free radical biology & medicine

    Article Title: Cys-141 glutathionylation of human p53: Studies using specific polyclonal antibodies in cancer samples and cell lines

    doi: 10.1016/j.freeradbiomed.2010.06.020

    Figure Lengend Snippet: Detection of glutathionylated p53 protein in human tumor cells using anti p53-glut antibodies Induction of glut-p53 protein was determined in HCT116 cells after 0.4 mM H 2 O 2 treatment for different times (A), after 20 min incubation with H 2 O 2 (0.4 mM), TBH (0.6 mM) and diamide (0.6 mM) (B), with camptothecin (2 μM, 5h) (C) , and with camptothecin (2 μM), cisplatin (10 μM), and doxorubicin (5 μM) for 6 and 12 h (C). In the last four lanes of (A) and (B) and last lane of (C) the protein samples were treated with DTT before SDS-PAGE to reverse p53 glutathionylation. C, untreated control; CPT, camptothecin.

    Article Snippet: The anticancer drugs cisplatin, doxorubicin and camptothecin were obtained from LKT laboratories (St. Paul, MN).

    Techniques: Incubation, SDS Page, Cycling Probe Technology

    Heatmap showing log 2 fold changes for treatments of staurosporine (STS), camptothecin (CPT) and copper oxide nanoparticles (CuO NPs) for the various time points ( n = 3), the levels of identification are indicated by 1 identified by reference substance (MS 2 and RT), 1a identified by reference substance (only RT), 2 identified by comparison of MS 2 spectra with reference databases such as Metlin. The dendrogram on the left hand side shows a clustering of similarly regulated metabolites upon treatment with STS/CPT/CuO NPs

    Journal: Particle and Fibre Toxicology

    Article Title: Copper oxide nanoparticle toxicity profiling using untargeted metabolomics

    doi: 10.1186/s12989-016-0160-6

    Figure Lengend Snippet: Heatmap showing log 2 fold changes for treatments of staurosporine (STS), camptothecin (CPT) and copper oxide nanoparticles (CuO NPs) for the various time points ( n = 3), the levels of identification are indicated by 1 identified by reference substance (MS 2 and RT), 1a identified by reference substance (only RT), 2 identified by comparison of MS 2 spectra with reference databases such as Metlin. The dendrogram on the left hand side shows a clustering of similarly regulated metabolites upon treatment with STS/CPT/CuO NPs

    Article Snippet: Staurosporine (STS) was purchased from Proteinkinase.de (Kassel, Germany), camptothecin (CPT) from Abcam (Cambridge, UK), and rhTNF-α from Immunotools (Friesoythe, Germany).

    Techniques: Cycling Probe Technology, Mass Spectrometry

    Apoptosis (caspase-3 and −7 activity) in response to CuO NPs, staurosporine (STS) and camptothecin (CPT). A549 cells treated with ( a ) CuO NPs at 0, 2.5, 5.0, 10 and 20 μg/ml, ( b ) STS at 0.5, 1, and 2 μM, or ( c ) CPT at 8, 16, and 32 μg/ml; for 3, 6, 12 and 24 h and assessed for caspase-3 and −7 activity. Data is expressed as % enzyme activity compared to medium only treated cells, and each data point represents the mean ± SEM ( n = 3). Statistical significance was determined by ANOVA with Tukey posthoc, and + for 6 h, ^ for 12 h and * for 24 h, when p

    Journal: Particle and Fibre Toxicology

    Article Title: Copper oxide nanoparticle toxicity profiling using untargeted metabolomics

    doi: 10.1186/s12989-016-0160-6

    Figure Lengend Snippet: Apoptosis (caspase-3 and −7 activity) in response to CuO NPs, staurosporine (STS) and camptothecin (CPT). A549 cells treated with ( a ) CuO NPs at 0, 2.5, 5.0, 10 and 20 μg/ml, ( b ) STS at 0.5, 1, and 2 μM, or ( c ) CPT at 8, 16, and 32 μg/ml; for 3, 6, 12 and 24 h and assessed for caspase-3 and −7 activity. Data is expressed as % enzyme activity compared to medium only treated cells, and each data point represents the mean ± SEM ( n = 3). Statistical significance was determined by ANOVA with Tukey posthoc, and + for 6 h, ^ for 12 h and * for 24 h, when p

    Article Snippet: Staurosporine (STS) was purchased from Proteinkinase.de (Kassel, Germany), camptothecin (CPT) from Abcam (Cambridge, UK), and rhTNF-α from Immunotools (Friesoythe, Germany).

    Techniques: Activity Assay, Cycling Probe Technology