Journal: The Journal of investigative dermatology

Article Title: Chloroquine Promotes Apoptosis in Melanoma Cells by Inhibiting BH3 domain Mediated PUMA Degradation

doi: 10.1038/jid.2013.56

Figure Lengend Snippet: a & b) Indicated melanoma cells were exposed to etoposide or different concentrations of CQ and the cell extracts were blotted with PUMA, p53, p21 and actin antibodies. c) Shows quantification of PUMA signal intensity from a & b. d & e) Melanoma cells were treated with etoposide or CQ and the relative levels of PUMA transcripts were assayed by q-PCR.

Article Snippet: Bafilomycin A, chloroquine diphosphate, leupeptin, and MG-132 were obtained from Sigma, ALLN, etoposide, and Z-Vad-Fmk from R&D Systems (Minneapolis), E64d and Pepstatin A (Cayman Chemical) and lactacystin (AG Scientific).

Techniques:

Journal: bioRxiv

Article Title: A developmentally programmed splicing failure attenuates the DNA damage response during mammalian zygotic genome activation

doi: 10.1101/2020.11.25.397794

Figure Lengend Snippet: (a) Working hypothesis for peak-like alternative exon regulation during ZGA in mouse. Left (Wild type): Snrpb / d2 have low mRNA and protein levels before ZGA. At ZGA, their mRNA levels are increased, followed by a rise in protein levels. This period with low levels of Snrpb / d2 protein leaves a time window in which sensitive exons in newly transcribed genes get excluded due to insufficient amount of these regulators. This situation starts being restored after ZGA, when Snrpb / d2 protein levels reach sufficient amounts. Given their specific functional enrichment, aberrant exclusion of exons in DDR genes during ZGA will contribute to a delay in the response to DNA damage at these developmental stages. Right: induced expression of Snrpb / d2 prior to ZGA should prevent the abovementioned exon skipping in DDR genes and therefore allow for an earlier stronger response to DNA damage. (b,c) Western blot and immunostaining showing SNRPB and SNRPBD2 levels during pre-implantation development in mouse. Pools of 150 or 60 embryos per stage were used for the SNRPB and SNRPD2 western blots respectively. (d) Changes in exon inclusion levels at 2C stage upon early induced expression of Snrpb/d2 (Y-axis) respect to the observed change at ZGA (2C-1C; X-axis). Exons with |ΔPSI| ≥ 15 upon Snrpb/d2 expression and at ZGA are highlighted in green/red and numbers indicated. p-value corresponds to a two-sided Binomial test between Q2+Q4 versus Q1+Q3. (e) Distribution of ΔPSI upon Snrpb/d2 induced expression for all exons with increased (UP), decreased (DOWN) or no change (NONE) in PSI at ZGA. (f) Pronuclear stage embryos were co-injected with Snrpb/d2 mRNA or injected with mCherry mRNA and left to develop to the 2C stage, when they were treated for 1.5h with 10uM etoposide or left untreated. Boxplots show the quantification of phospho-p53 (Ser15) relative intensity from immunostaining of embryos in each condition. Each dot represents the average relative intensity of both cells of a single embryo. Data from three independent experiments is shown (individual experiments are shown in Extended Data Fig. 10a). Total number of embryos for each condition: n=29 (mCherry untreated), n=26 (Snrpb/d2, untreated), n=30 (mCherry, etoposide) and n=29 (Snrpb/d2, etoposide). ** P = 0.0034, two-sided Wilcoxon Rank Sum test.

Article Snippet: For experiments in , embryos were either left untreated or treated for 1h at the stated stage with 0.5uM etoposide (Sigma).

Techniques: Functional Assay, Expressing, Western Blot, Immunostaining, Injection

Journal: bioRxiv

Article Title: A developmentally programmed splicing failure attenuates the DNA damage response during mammalian zygotic genome activation

doi: 10.1101/2020.11.25.397794

Figure Lengend Snippet: (a) Scheme of etoposide treatment and analysis of embryo recovery: 2C embryos (1.5 days post coitum, dpc) were either left un-treated or treated with 0.5uM etoposide for 1h and then washed and left to recover for further 48h. (b,c) Proportion of embryos found arrested or developed to the stated stage 24h (b) or 48h (c) post-treatment. Stack plots show the average of 6 independent experiments with a total number of embryos analysed of n=105 (control) and n=113 (treated). All embryos not reaching the expected developmental stage at a given time point either because of cell death or cell cycle arrest were classified as “Arrested”. (d) TUNEL staining of embryos in (b) and (c), showing a high incidence of cell death in treated embryos only 48h, but not 24h, post-etoposide treatment. (e) Percentage of embryos showing positive TUNEL staining at each developmental stage. Average of three independent experiments is shown with a total number of embryos of: n=72 (control, 2.5dpc), n=63 (etoposide, 2.5dpc), n=67 (control, 3.5dpc) and n=50 (etoposide, 3.5dpc). * P =0.036 two-sided Student’s T-test. (f,g) Immunostaining for phospho-p53 (Ser15; pp53) in 2C (f) or early morula (g) stage embryos treated with 0.5uM etoposide for 1h or left untreated. Quantification of phospho-p53 (Ser15) levels from embryos is shown for each stage. Each dot represents the average relative intensity of all cells of a single embryo. Boxplots show staining levels of embryos from three independent experiments. Number of embryos: n=42 (control, 2C), n=47 (etoposide, 2C), n=17 (control, morula) and n=25 (etoposide, morula). *** (0.001 < P ) based on Wilcoxon Rank Sum tests.

Article Snippet: For experiments in , embryos were either left untreated or treated for 1h at the stated stage with 0.5uM etoposide (Sigma).

Techniques: TUNEL Assay, Staining, Immunostaining

Journal: PLoS ONE

Article Title: TRAIL Receptor Signaling Regulation of Chemosensitivity In Vivo but Not In Vitro

doi: 10.1371/journal.pone.0014527

Figure Lengend Snippet: Isogenic BJAB cell lines were treated with increasing doses of etoposide, MS-275, oxamflatin, doxorubicin, MG132, UV, temozolomide, 5-FU, staurosporine or sorbitol as indicated followed by MTT assay to assess cell viability. All dose response curves overlap for each stimulus.

Article Snippet: The following drugs were prepared according to manufacturer's instructions and were applied in serial dilution format: TRAIL (R&D Systems, Minneapolis, MN), SuperFas Ligand (Enzo Life Sciences, Plymouth Meeting, PA), 5-Fluorouracil (5-FU), Doxorubicin Hydrochloride, Etoposide, Oxamflatin, Temozolomide, Sorbitol, MS-275, and Staurosporine (Sigma, St. Louis, MO), MG132 (EMD Biosciences, Gibbstown, NJ).

Techniques: MTT Assay

Journal: PLoS ONE

Article Title: TRAIL Receptor Signaling Regulation of Chemosensitivity In Vivo but Not In Vitro

doi: 10.1371/journal.pone.0014527

Figure Lengend Snippet: Isogenic control or FADD-DD expressing BJAB cells were treated with TRAIL or etoposide as indicated for 24 hours, then washed and replaced into growth media. Long term growth of surviving cells was determined by counting viable cells. Control BJAB cells died rapidly and were unable to recover any long term growth. Etoposide treated cells were completely unable to recover growth capacity whether or not FADD-DD was expressed. However FADD-DD expression protected the TRAIL-treated cells as demonstrated by overlapping growth curves with the untreated controls.

Article Snippet: The following drugs were prepared according to manufacturer's instructions and were applied in serial dilution format: TRAIL (R&D Systems, Minneapolis, MN), SuperFas Ligand (Enzo Life Sciences, Plymouth Meeting, PA), 5-Fluorouracil (5-FU), Doxorubicin Hydrochloride, Etoposide, Oxamflatin, Temozolomide, Sorbitol, MS-275, and Staurosporine (Sigma, St. Louis, MO), MG132 (EMD Biosciences, Gibbstown, NJ).

Techniques: Control, Expressing

Journal: PLoS ONE

Article Title: TRAIL Receptor Signaling Regulation of Chemosensitivity In Vivo but Not In Vitro

doi: 10.1371/journal.pone.0014527

Figure Lengend Snippet: HCT-116 cells transfected with GFP, FADD-DD or FADD-DDV108E expression constructs were treated with increasing doses of TRAIL, FasL, etoposide, 5-FU, or doxorubicin as indicated and cell viability was assessed. FADD-DD and FADD-DDV108E inhibited FasL and TRAIL as in BJAB cells, but had no effect on tumor cell killing by the chemotherapy drugs.

Article Snippet: The following drugs were prepared according to manufacturer's instructions and were applied in serial dilution format: TRAIL (R&D Systems, Minneapolis, MN), SuperFas Ligand (Enzo Life Sciences, Plymouth Meeting, PA), 5-Fluorouracil (5-FU), Doxorubicin Hydrochloride, Etoposide, Oxamflatin, Temozolomide, Sorbitol, MS-275, and Staurosporine (Sigma, St. Louis, MO), MG132 (EMD Biosciences, Gibbstown, NJ).

Techniques: Transfection, Expressing, Construct

Journal: PLoS ONE

Article Title: TRAIL Receptor Signaling Regulation of Chemosensitivity In Vivo but Not In Vitro

doi: 10.1371/journal.pone.0014527

Figure Lengend Snippet: Panel A, isogenic BJAB cells expressing GFP control, GFP-FADD-DD and GFP-FADD-DDV108E were implanted subcutaneously and tumors grown for 10 days prior to treatment with etoposide. Untreated tumors continued to grow. In control BJAB cells, etoposide caused tumor eradication; whereas, in tumors expressing either FADD-DD or FADD-DDV108E, etoposide treatment led to stabilization of tumor mass but no eradication (p<0.05 by t-test at 18 for the control versus FADD-DD and FADD-DDV108E expressing cells). Panel B, Western blot of tumor tissue from GFP control, GFP-FADD-DD and GFP-VFADD-DD V108E demonstrating similar expression of the GFP-tagged protein in all tumors.

Article Snippet: The following drugs were prepared according to manufacturer's instructions and were applied in serial dilution format: TRAIL (R&D Systems, Minneapolis, MN), SuperFas Ligand (Enzo Life Sciences, Plymouth Meeting, PA), 5-Fluorouracil (5-FU), Doxorubicin Hydrochloride, Etoposide, Oxamflatin, Temozolomide, Sorbitol, MS-275, and Staurosporine (Sigma, St. Louis, MO), MG132 (EMD Biosciences, Gibbstown, NJ).

Techniques: Expressing, Control, Western Blot

Journal: PLoS ONE

Article Title: TRAIL Receptor Signaling Regulation of Chemosensitivity In Vivo but Not In Vitro

doi: 10.1371/journal.pone.0014527

Figure Lengend Snippet: Panel A, parental BJAB cells and BJAB cells expressing Six1 were treated in vitro with increasing doses of TRAIL, FasL and etoposide, and cell viability was assessed by the MTT assay. Six1 caused TRAIL resistance but had little effect on FasL or etoposide-induced cell death. Panel B illustrates how etoposide treatment of subcutaneous tumors from BJAB or BJAB-Six1 cells reduces the growth of both tumors relative to untreated controls, but is less effective in the Six1-expressing cells (* p<0.001 by t-test at day 14 for the control versus Six1 expressing cells).

Article Snippet: The following drugs were prepared according to manufacturer's instructions and were applied in serial dilution format: TRAIL (R&D Systems, Minneapolis, MN), SuperFas Ligand (Enzo Life Sciences, Plymouth Meeting, PA), 5-Fluorouracil (5-FU), Doxorubicin Hydrochloride, Etoposide, Oxamflatin, Temozolomide, Sorbitol, MS-275, and Staurosporine (Sigma, St. Louis, MO), MG132 (EMD Biosciences, Gibbstown, NJ).

Techniques: Expressing, In Vitro, MTT Assay, Control

Journal: iScience

Article Title: Borna disease virus docks on neuronal DNA double-strand breaks to replicate and dampens neuronal activity

doi: 10.1016/j.isci.2021.103621

Figure Lengend Snippet: BoDV-1 increases levels of DNA double-strand breaks in primary neurons (A) Schematic representation of the experimental strategy using primary cultures of rat hippocampal neurons and BoDV-1 infection. (B) Insets views are displayed from confocal microscopy analysis of 53BP1 staining (red) and MAP2-positive (blue) neurons from BoDV-1 (BoDV) versus non-infected (NI) cultures. Arrows indicate 53BP1-positive foci of DSB. Scale bar: 10 μm. (C) Numbers of neurons with 53BP1-positive foci were counted and are expressed as average percentages of foci-positive neurons. A total of 2,358 NI- and 2,697 BoDV-1 infected neurons were observed in n = 2–3 fields on 4–6 coverslips per condition from 3 independent experiments. ∗∗∗p = 0.001 versus NI by unpaired t test. (D-E) Levels of the DSB marker γH2A.X were determined by Western blotting. (D) A Western blot illustrating particularly strong increase in γH2A.X signals upon infection. Nucleoprotein (N) and β-actin levels were also detected. (E) Quantitation of Western blot signals. The average γH2A.X to β-actin ratio in non-infected cultures was defined as 1.0. n = 2–6 wells per condition from 7 independent experiments. ∗∗∗∗p < 0.0001 versus NI by unpaired t test with Welch correction. (F-I) Cell nuclei isolated from 14-DIV primary mixed cultures of hippocampal neuron homogenates were assessed for DNA fragmentation levels by the comet assay at neutral pH. (F) Representative images of cell nuclei from non-infected (NI), BoDV-infected (BoDV) cultures or as a positive control, after 5 h incubation with 0.5 μM Etoposide (ETP). Images were captured by fluorescence microscopy. Scale bar: 10 μm. (G) Quantification of the percentage of nuclei with comet tails, which are indicative of DSB. For each condition, a total of 1,325–2,190 nuclei were inspected and scored. (H) Tail moment, indicating the severity of DNA fragmentation as a function of the number of fragments produced and the lengths of the fragments, was measured for each cell showing a comet. (I) Tail length, indicating the extent of DNA fragmentation, was measured for each cell showing a comet. Tail moment and length are expressed as normalized on the average tail moment/length for the NI measured for each experiment. n = 6–8 wells per condition from 4 independent experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 versus leftmost bar (Dunnett post hoc test). Bars represent means ± SEM.

Article Snippet: To achieve global activation of N-methyl-D-aspartate receptors (NMDAR), after washing cells with 1X PBS at 37°C, neurons were stimulated with 15 μM NMDA (Sigma) in prewarmed conditioned media during 30 min. To study the link between DSB and viral replication, infected neurons were treated with various concentration of Etoposide (ETP; Sigma) in DMSO, 2 days post-infection (dpi) during 5 days (vSPOT and DSB analysis), or treated 5 dpi during 24, 48, 72 hours (viral RNA (vRNA) analysis).

Techniques: Infection, Confocal Microscopy, Staining, Marker, Western Blot, Quantitation Assay, Isolation, Single Cell Gel Electrophoresis, Positive Control, Incubation, Fluorescence, Microscopy, Produced

Journal: iScience

Article Title: Borna disease virus docks on neuronal DNA double-strand breaks to replicate and dampens neuronal activity

doi: 10.1016/j.isci.2021.103621

Figure Lengend Snippet: Induction of DSB in neurons by Etoposide increases numbers of colocalized DSB/vSPOT per neuron and viral RNA replication (A) Schematic representation of the experimental strategy. Primary cultures of rat hippocampal neurons were infected by BoDV-1 and treated with the DSB-inducing agent Etoposide (ETP) or its vehicle DMSO, followed by immunofluorescence analysis. (B–D) The numbers of DSB and vSPOT and the levels of colocalization of these two nuclear events were assessed by manual counting following immunofluorescence staining of 53BP1-positive foci of DSB (in red), and Nucleoprotein-positive (N) vSPOT (in green) analyzed by confocal microscopy. (B) Representative confocal images of DSB foci, vSPOT, and their colocalization (arrow), for experiments using 0.1 or 0.5 μM Etoposide, or vehicle-treated (DMSO) cultures. Scale bar: 10 μm. (C) Distribution as percentage of the counts of infected neurons displaying DSB foci in three categories (1, 2, or ≥3 DSB/cell) depending on the numbers of foci per nucleus observed. (D) Distribution of the counts of the same neurons displaying vSPOT in three categories (1, 2, or ≥3 vSPOT/cell) depending on the numbers of events per nucleus observed. n = 22–44 neurons analyzed from 2 coverslips per condition, from 3 independent experiments. ns, non-significant, ∗∗∗∗p < 0.0001 by multiple t test. (E and F) Quantitative analysis by RT-qPCR of BoDV-1 viral RNA levels in rat hippocampal neurons, after infection by BoDV-1 and at 3 different times post treatment with Etoposide (ETP) or vehicle (DMSO). (E) Schematic representation of the experimental strategy. (F) Changes in RNA levels are expressed as compared with the DMSO condition, for which the average ΔΔCt (normalized on GAPDH) was set to 1. n = 2 wells analyzed each in duplicates, per condition from 4 independent experiments. ns, non-significant, ∗∗p < 0.01 by Bonferroni post hoc t test. Bars represent means ± SEM.

Article Snippet: To achieve global activation of N-methyl-D-aspartate receptors (NMDAR), after washing cells with 1X PBS at 37°C, neurons were stimulated with 15 μM NMDA (Sigma) in prewarmed conditioned media during 30 min. To study the link between DSB and viral replication, infected neurons were treated with various concentration of Etoposide (ETP; Sigma) in DMSO, 2 days post-infection (dpi) during 5 days (vSPOT and DSB analysis), or treated 5 dpi during 24, 48, 72 hours (viral RNA (vRNA) analysis).

Techniques: Infection, Immunofluorescence, Staining, Confocal Microscopy, Quantitative RT-PCR

Journal: iScience

Article Title: Borna disease virus docks on neuronal DNA double-strand breaks to replicate and dampens neuronal activity

doi: 10.1016/j.isci.2021.103621

Figure Lengend Snippet:

Article Snippet: To achieve global activation of N-methyl-D-aspartate receptors (NMDAR), after washing cells with 1X PBS at 37°C, neurons were stimulated with 15 μM NMDA (Sigma) in prewarmed conditioned media during 30 min. To study the link between DSB and viral replication, infected neurons were treated with various concentration of Etoposide (ETP; Sigma) in DMSO, 2 days post-infection (dpi) during 5 days (vSPOT and DSB analysis), or treated 5 dpi during 24, 48, 72 hours (viral RNA (vRNA) analysis).

Techniques: Recombinant, Transfection, Protease Inhibitor, Blocking Assay, SYBR Green Assay, TUNEL Assay, In Situ, Plasmid Preparation, Software

Journal: Scientific Reports

Article Title: Transactivation of human osteoprotegerin promoter by GATA-3

doi: 10.1038/srep12479

Figure Lengend Snippet: ( A ) HEK cells expressing either a scrambled shRNA or a GATA-3 shRNA expressing plasmid were treated with etoposide (10 μM), and apoptosis was assayed by Western blot (left). HEK cells expressing a GATA-3 shRNA expressing plasmid were treated with purified OPG protein for 2 h before treatment with etoposide, and apoptosis was assayed by Western blot (right). Full-length blots are presented in . ( B ) HEK cells expressing either a control plasmid or a GATA-3 shRNA expressing plasmid were treated with TNF-α (2 μg/mL) with or without pre-treatment with OPG for 2 h. Subsequent cell death was measured using TUNEL assays (left), and quantified with bar graphs (right). *p < 0.05, n = 3.

Article Snippet: The apoptosis inducing agents, tumor necrosis factor alpha (TNF-α) (#8902) and etoposide (#341205), were from Cell Signaling and EMD Millipore (USA), respectively.

Techniques: Expressing, shRNA, Plasmid Preparation, Western Blot, Purification, Control, TUNEL Assay

Journal: Oncotarget

Article Title: Cartilage oligomeric matrix protein promotes prostate cancer progression by enhancing invasion and disrupting intracellular calcium homeostasis

doi: 10.18632/oncotarget.21176

Figure Lengend Snippet: ER stress-mediated apoptosis was induced in the transfected cells with Brefeldin A. Cells expressing COMP were less apoptotic as compared to mock (A) . Similar results were obtained when apoptosis was induced via the TNFR1 pathway (B) . Apoptosis was, then, induced by inhibiting protein translation (CHX, cycloheximide), RNA synthesis (actinomycin D), protein kinases (staurosporine) or nuclear topoisomerase (camptothecin and etoposide). COMP expression was protective against all apoptotic stimuli except inhibition of nuclear topoisomerase (C) . Furthermore, COMP expression protected the cells against apoptosis induced by Docetaxel (D) . Addition of recombinant COMP to mock-trasfected cells could not protects the cells against apoptosis induced by staurosporin, suggesting that the protective effect is mediated by intracellular COMP (E) . The data represent at least three independent experiments ±SD and was compared to mock by 2-way ANOVA with Bonferroni post-test (A-D), or compared to BSA by 1-way ANOVA with Dunnett's multiple comparison test (E), ns, not significant; * , p < 0.05; ** , p < 0.005; *** , p < 0.001.

Article Snippet: In addition, apoptosis was induced with 10 μM Camptothecin, 100 μM CHX, 10 μM Actinomycin D, 100 μM Etoposide (Apoptosis inducer set I, Calbiochem), or 1 μM Staurosporine (Sigma-Aldrich).

Techniques: Transfection, Expressing, Inhibition, Recombinant, Comparison

Journal: Oncotarget

Article Title: Cartilage oligomeric matrix protein promotes prostate cancer progression by enhancing invasion and disrupting intracellular calcium homeostasis

doi: 10.18632/oncotarget.21176

Figure Lengend Snippet: Cells expressing COMP have decreased oxygen consumption rate (OCR), reflecting OXPHOS (A) with a concurrent upregulation of lactate production by glycolysis (B) , which indicates an enhanced Warburg effect. Ca 2+ release into the cytosol after stimulation with glucose is abolished in cells expressing COMP, as was shown by live cell imaging (C) and flow cytometry (D) . To show that the Ca 2+ is released from the ER and not taken up from the buffer, cytosolic Ca 2+ levels were monitored upon glucose or thapsigargin treatment in a buffer lacking Ca 2+ . A lower basal cytosolic Ca 2+ level was observed in cells expressing COMP, as well as no Ca 2+ release upon treatment (E) . In addition, live imaging experiments were performed under conditions without Ca 2+ . Mock-transfected cells had a significantly higher uptake of extracellularly added Ca 2+ as compared to COMP-transfected cells (F) . In further support, no free Ca 2+ was taken up by the mitochondria of COMP-transfected cells upon stimulation with histamine (G) . Inhibition of ER Ca 2+ release, with 2-APB, abolished the differences in apoptosis between mock- and COMP-transfected cells (H) , suggesting that the anti-apoptotic effect of COMP is connected to impaired Ca 2+ signalling. Each graph represents three independent experiments and the data were analysed by 2-way ANOVA with Bonferroni post-test (A, C, E-F, H) or 2-tailed student's t-test (B, D, G) comparing to mock-transfected cells. The symbols ns, * , ** , and *** stand for not significant, p < 0.05, p < 0.005 and p < 0.001, respectively.

Article Snippet: In addition, apoptosis was induced with 10 μM Camptothecin, 100 μM CHX, 10 μM Actinomycin D, 100 μM Etoposide (Apoptosis inducer set I, Calbiochem), or 1 μM Staurosporine (Sigma-Aldrich).

Techniques: Expressing, Live Cell Imaging, Flow Cytometry, Imaging, Transfection, Inhibition