etoposide Search Results


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  • 99
    Millipore etoposide
    Scheme of Hsp27-induced effects on apoptosis. Hsp27 reduces F-actin damage induced by apoptotic drugs (e.g., cytochalasin D and staurosporine) and thus attenuates the activation of the pathway that links F-actin damages to mitochondria. Activation of this pathway induces cytochrome c release, apoptosome formation, and procaspase activation. The mechanism of activation of this pathway is unknown but may be a consequence of altered integrin signaling pathway or changes in F-actin-dependent subcellular distribution of members of the Bcl-2 family such as Bid. Hsp27 also attenuates cytochrome c release in cells exposed to agents that do not rapidly destroy F-actin architecture (e.g., <t>etoposide</t> and Fas), suggesting that other upstream pathways are under the control of Hsp27 expression. Hsp27 also acts downstream of mitochondria by interfering with apoptosome formation, probably through its binding to cytochrome c once it is released from mitochondria. Hsp27 also appears to bind and negatively modulate caspase 3. In L929 cells, the upstream activity necessitates a higher level of Hsp27 expression ( > 0.45 ng/μg) compared to the downstream effect which is already detected in cells expressing Hsp27 (0.1 ng/μg).
    Etoposide, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8224 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Selleck Chemicals etoposide
    HT1080 FUCCI show strong cell cycle-associated cell death. Cell cycle-associated death standards are used. ( a ) A representative FUCCI trace of cells treated with the topoisomerase-α poison, <t>etoposide.</t> Cells progress from G1-phase (red), with normal kinetics, progress to a green state and die, consistent with S/G2-phase associated death. ( b ) A representative FUCCI trace of cells treated with the DNA modifier, cisplatin. Cells most often progress normally from G1-phase (red) to an all green state and die, consistent with S-phase associated death. ( c ) A representative FUCCI trace of cells treated with a Kinesin-5 inhibitor, K5I. This cell progresses through the cell cycle with normal kinetics and enters mitotic arrest at 14 h post-treatment (*). While arrested, red signal is reacquired after 3–4 h, beginning at 17 h. This cell dies at 23 h and nearly all other cells also die while arrested in mitosis. Arrows indicate time of death. See Supplementary Fig. 1e–g online for FUCCI distributions over time. Supplementary video S6-8 online. Cell number tracked: etoposide, 33, cisplatin, 21, K5I, 30.
    Etoposide, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 99/100, based on 192 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Bristol Myers vepesid
    <t>Etoposide</t> and ANS-etoposide toxicity in GSTA1 and MGST1 overexpressing cells. Cellular toxicity of (A) etoposide and (B) ANS–etoposide analyzed with the MTT assay after short-term 24 h exposure with indicated doses, in at least three independent experiments measured in at least triplicate (mean ± SEM, n ≥ 9, * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001). Left panel: MGST1 overexpressing MCF7 cells (dark gray/MGST1) and the vector control (light gray/control). Right panel: GSTA1 overexpressing V79 cells (dark gray/GSTA1) and the vector control (light gray/control).
    Vepesid, supplied by Bristol Myers, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Enzo Biochem etoposide
    HDAC4 is dysregulated during senescence and aging and is required for senescence escape. a. Immunoblot analysis in IMR90 cells undergoing replicative senescence. Actin was used as loading control. b. Microscopic images of SA-β-gal stained IMR90 cells (scale bar 50 µm). c. Immunoblot analysis in tissue-derived lysates obtained from C57BL/6J female mice sacrificed at 128 (young) and 774 (old) days of age. Actin was used as loading control. d. Immunoblot analysis in BJ/ hTERT expressing the indicated transgenes for the indicated time. Vimentin was the loading control. e. Cellular lysates obtained in BJ/ hTERT expressing for 8 days the indicated transgenes and treated or not for 8h with MG132 were immunoprecipitated using anti-HDAC4 and immunoblotted with the indicated antibodies. f. Immunoblot analysis in BJ/ hTERT cells expressing HRAS G12V and silenced for GSK3β, as indicated. g. Immunoblot analysis in BJ/ hTERT/E1A/RAS / HDAC4 + / + or -/- cells, as indicated. Actin was used as loading control. h. Analysis of the senescent cells as scored after SA-β-gal staining. Mean ± SD; n = 3. i. Immunoblot analysis in SK-LMS-1/ HDAC4 + / + or -/- as indicated. j. Cell-proliferation curve of the indicated HDAC4 + / +, + /-, -/- SK-LMS-1 cells. Mean ± SD; n = 4. k. Analysis of the senescent cells as scored after SA-β-gal staining. Mean ± SD; n = 4. l. Representative image of normal and altered DAPI-stained nuclei observed in SK-LMS-1 HDAC4 -/- cells (scale bar 10 µm). m-n. Analysis of the % of cells displaying altered nuclei or γH2AX foci ( > 5). <t>Etoposide</t> was a control (2h, 20µM). Mean ± SD; n = 4. o-p-q. Analysis of SA-β-gal (o), BrdU (p) and γH2AX (q) positivity in wt or HDAC4 KO cells expressing HYGRO R (clone 635) or HDAC4 PAM . Mean ± SD; n = 4. The significance is relative to clone 635. r. Immunoblot analysis in SK-LMS-1 wt or KO (clone 66) cells re-expressing a tamoxifen inducible HDAC4 PAM -ER . Arrowheads point to HDAC4 cleavage products observed in HDAC4-ER expressing cells. Actin was the loading control. s-v. Analysis of SA-β-gal (s), BrdU (t), nuclear alteration (u) and γH2AX (v) positivity in the indicated cells. Mean ± SD; n = 4.
    Etoposide, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 93/100, based on 252 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Teva etoposide
    A viability metric reports overall dose response and heterogeneity based on relative esterase activity and membrane permeability. In (a), representative thumbnails from merged images of esterase-cleaved calcein AM (green) and intercalated ethidium bromide (red) in ovarian cancer 3D overlays for (left to right), no treatment (NT) and total killing (TK) and increasing doses of paclitaxel (as a representative cytotoxic agent). Thumbnails qualitatively display increasing ethidium bromide intercalation (red) and decreasing esterase activity (green) with increasing cytotoxic dose similar for all therapies assayed here. Scale bars = 500 μm. In (b), mean fluorescence signals from full fields after rescaling ( Equation 1 ) are shown for escalating doses of paclitaxel, carboplatin, <t>etoposide,</t> and verteporfin PDT treatments (from left to right) in the upper plots used to quantify overall viability (lower plots, from Equation 2 ) and estimate fractional lethal doses (LD). In (c), distributions of nodule viabilities ( N > 1000 ) from automated segmentation (shown for paclitaxel-treated cultures) shifts to the left as expected with increasing dose. In (d), paclitaxel and carboplatin viability distributions at LD80 (thumbnails, left, scalebars = 500 μm) are juxtaposed to reveal a relatively tight distribution in the former in contrast to heterogeneous viability spectrum in the latter with sub-populations up to 80–90% viable.
    Etoposide, supplied by Teva, used in various techniques. Bioz Stars score: 92/100, based on 148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Cell Signaling Technology Inc etoposide
    p53 induction in response to DNA damage is impaired in TFEB/TFE3 DKO RAW264.7. 7 cells. ( A ) Representative Western blot showing p53 induction, p53 Ser15 phosphorylation, and Mdm2 levels in WT and TFE3/TFEB DKO RAW264.7 cells following <t>etoposide</t> treatment up to 8 hr. ( B ) Quantification of p53 induction from data shown in A. Data represents mean relative p53 level ± standard deviation with n = 3. Significance tested with two-way ANOVA with Sidak’s multiple comparisons test (**p
    Etoposide, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 100 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cayman Chemical etoposide
    Association of CFI-400945 with conventional cytotoxic drugs doxorubicin and <t>etoposide</t> synergistically reduced viability of RT and MB cell lines 5nM and 10nM CFI-400945 were used in combination with doxorubicin and etoposide in concentrations ranging from 0.001 to 5μM. Viability was assessed comparing the effects of combination therapy versus treatment with each cytotoxic drug alone. Median effect plots showed that CFI-400945 in association with etoposide or doxorubicin significantly decreased the concentration of cytotoxic drug needed to affect viability of RT and MB cells (for IC50 values, please refer to Table 1 ).
    Etoposide, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 92/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    BioVision etoposide
    The lncRNA transcription rates were not affected by chemical stressors. The transcription rates of the lncRNAs ( a ) OIP5-AS1, ( b ) FLJ46906, ( c ) LINC00137, and ( d ) GABPB1-AS1 were examined in control cells (black bar) and those exposed to hydrogen peroxide (gray bar), mercury II chloride (pale gray bar), or <t>etoposide</t> (white bar) as chemical stressors. The nascent lncRNAs incorporated EU during transcription, and the relative EU-RNA quantity reflects the total amount of EU-labeled RNA captured divided by the input amount of RNA as an indicator of the transcription rate. All values are means ± SD from three independent experiments.
    Etoposide, supplied by BioVision, used in various techniques. Bioz Stars score: 93/100, based on 55 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Bedford Laboratories etoposide
    Perifosine increased the sensitivity of high TrkB expressing TB3 tumors to <t>etoposide</t> treatment. A. Mice were treated with vehicle or perifosine at 10mg/kg, 15mg/kg or 20mg/kg for 30 days. The tumor size in treated groups was compared to that of control group. # P
    Etoposide, supplied by Bedford Laboratories, used in various techniques. Bioz Stars score: 92/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Tocris etoposide
    p53 R175H and MDM2 proteins synergistically reduce chemosensitivity of lung and breast cancer cells ( A ) H1299-R175H-MDM2 cell line was treated with Ponasterone A (Pon A) and/or Doxycycline (Dox) for 24 h to induce p53 R175H and/or MDM2, respectively. Immunoblotting with specific antibody revealed tight and efficient expression of both proteins. ( B ) Induced and uninduced cells were grown in triplicate in chambers compatible with the xCELLigence RTCA DP Instrument and Cisplatin (40 μM) was added at the indicated time point. Proliferative index was monitored for 120 h. Mean and standard deviation of three repeats are shown. ( C ) After 48 h treatment with 60 μM Cisplatin (left panel) or <t>Etoposide</t> (right panel), the apoptotic response of induced or uninduced cells stained with Annexin V/Gel Green dye was measured with a flow cytometer. p53 R175H or MDM2 expressed alone reduced apoptosis to same extent, whereas significant decrease was observed after simultaneous induction of both proteins. Bars represent the relative decrease (%) of cells in early apoptosis (Annexin V positive, Gel Green dye negative), estimated as follows = ( Value − Baseline ) Baseline × 100 (Baseline–Apoptotic response of uninduced H1299-R175H-MDM2 cell line). Statistical significance ( P value) was counted for three independent experiments with Anova statistical test. *, **, ***, **** indicate statistical significance p
    Etoposide, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam etoposide
    DNA damage induces de-phosphorylation of nuclear Tau. ( a ) The effect of <t>Etoposide</t> and Vinblastine treatment on mouse C17.2 cells is shown by confocal microscopy upon immune staining of PFA-fixed cells with antibodies against the microtubule marker β-tubulin (in cyan) or the DNA damage marker γH2A-X (in red). ( b ) Confocal microscopic quantification of the activated kinases in the nucleus (DAPI mask). Mean percent ± sem relative to the respective controls. 2-tailed unpaired Mann-Whitney test, ****p
    Etoposide, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    FUJIFILM etoposide
    Characteristics of H1299 and A549  VR  cells. (A) H1299 parental and  VR  (weak, moderate, and strong resistant) cell viabilities in response to  VRB . H1299 parental and  VR  cells were treated for 96 h with increasing concentrations of  VRB . Cell viability was determined using the  WST ‐8 assay and is shown as a percentage of the value of the untreated cells. The sigmoid curves were drawn using Prism software. Experiments were performed in triplicate. (B) The  IC 50 values for  VRB ,  PAC ,  DOC ,  VP ‐16, and  CDDP  in H1299 parental and  VR  cells. Cell viability in response to  PAC ,  DOC ,  VP ‐16, and  CDDP  was measured as described for  VRB . Experiments were performed in triplicate. The  IC 50 values were calculated using Prism software. The error bars show the 95%  CI . *  P
    Etoposide, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 93/100, based on 96 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Amaxa etoposide treatment
    <t>Etoposide-induced</t> DSB repair by NHEJ involves the MRN complex. γH2AX foci kinetics were assessed in primary human fibroblasts. Mre11-defective (ATLD2) and Nbs1-defective (CZD82CH and GM07166A) but not ATM-defective primary human fibroblasts (AT1BR) exhibit elevated foci levels after 20 µM etoposide treatment in G0/G1 phase. Background foci numbers were subtracted. Error bars represent the SD from at least three different experiments.
    Etoposide Treatment, supplied by Amaxa, used in various techniques. Bioz Stars score: 89/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pharmachemie etoposide
    Sensitivity of primary and transformed cells to DNA damaging agents in the presence and absence of C9orf82 protein. (A) Relative survival of pre-B cells from wild type and C9orf82 ko/ko mice upon exposure to increasing doses of UVC, cisplatin, MMS, γ-irradiation, doxorubicin and <t>etoposide</t> was analyzed. (B) Colony survival rate of transformed MEFs from wild type and C9orf82 ko/ko mice upon exposure to Doxorubicin and Etoposide was compared. (C) Recovery from DNA damage response. The recovery rate from γH2AX generated in Tp53kd transformed MEFs in response to etoposide induced DSBs are compared. (D) Cell cycle analysis and relative contribution of sub-G, G1, and S/G2 of pre-B cells from the indicted genotypes.
    Etoposide, supplied by Pharmachemie, used in various techniques. Bioz Stars score: 92/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biomol GmbH etoposide
    Tipifarnib enhances induction of apoptosis by <t>etoposide</t> in AML cell lines . (A) Log-phase HL-60 cells were treated for 24 hours with diluent, 1.5 μM etoposide, 1 μM tipifarnib, or the combination of 1.5 μM etoposide plus 1 μM
    Etoposide, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Scheme of Hsp27-induced effects on apoptosis. Hsp27 reduces F-actin damage induced by apoptotic drugs (e.g., cytochalasin D and staurosporine) and thus attenuates the activation of the pathway that links F-actin damages to mitochondria. Activation of this pathway induces cytochrome c release, apoptosome formation, and procaspase activation. The mechanism of activation of this pathway is unknown but may be a consequence of altered integrin signaling pathway or changes in F-actin-dependent subcellular distribution of members of the Bcl-2 family such as Bid. Hsp27 also attenuates cytochrome c release in cells exposed to agents that do not rapidly destroy F-actin architecture (e.g., etoposide and Fas), suggesting that other upstream pathways are under the control of Hsp27 expression. Hsp27 also acts downstream of mitochondria by interfering with apoptosome formation, probably through its binding to cytochrome c once it is released from mitochondria. Hsp27 also appears to bind and negatively modulate caspase 3. In L929 cells, the upstream activity necessitates a higher level of Hsp27 expression ( > 0.45 ng/μg) compared to the downstream effect which is already detected in cells expressing Hsp27 (0.1 ng/μg).

    Journal: Molecular and Cellular Biology

    Article Title: Hsp27 as a Negative Regulator of Cytochrome c Release

    doi: 10.1128/MCB.22.3.816-834.2002

    Figure Lengend Snippet: Scheme of Hsp27-induced effects on apoptosis. Hsp27 reduces F-actin damage induced by apoptotic drugs (e.g., cytochalasin D and staurosporine) and thus attenuates the activation of the pathway that links F-actin damages to mitochondria. Activation of this pathway induces cytochrome c release, apoptosome formation, and procaspase activation. The mechanism of activation of this pathway is unknown but may be a consequence of altered integrin signaling pathway or changes in F-actin-dependent subcellular distribution of members of the Bcl-2 family such as Bid. Hsp27 also attenuates cytochrome c release in cells exposed to agents that do not rapidly destroy F-actin architecture (e.g., etoposide and Fas), suggesting that other upstream pathways are under the control of Hsp27 expression. Hsp27 also acts downstream of mitochondria by interfering with apoptosome formation, probably through its binding to cytochrome c once it is released from mitochondria. Hsp27 also appears to bind and negatively modulate caspase 3. In L929 cells, the upstream activity necessitates a higher level of Hsp27 expression ( > 0.45 ng/μg) compared to the downstream effect which is already detected in cells expressing Hsp27 (0.1 ng/μg).

    Article Snippet: Crystal violet, staurosporine, cytochalasin D, phalloidin, etoposide, and l -buthionine-( S , R )-sulfoximine (BSO) were purchased from Sigma.

    Techniques: Activation Assay, Expressing, Binding Assay, Activity Assay

    Hsp27 expression interferes with the staurosporine- and etoposide-induced Bid intracellular relocalization in murine L929 cells. Control L929-C2 and Hsp27-expressing L929-Hsp27 cells kept either untreated (NT) or exposed to staurosporine (1 μM) or etoposide (500 μM) for either 1, 2, or 4 h were lysed under conditions which preserve mitochondrial and membrane integrity as described in Materials and Methods. The resulting P20 and S cytosolic fractions were analyzed in immunoblots probed with anti-Bid antibody. Autoradiographs of immunoblots are shown.

    Journal: Molecular and Cellular Biology

    Article Title: Hsp27 as a Negative Regulator of Cytochrome c Release

    doi: 10.1128/MCB.22.3.816-834.2002

    Figure Lengend Snippet: Hsp27 expression interferes with the staurosporine- and etoposide-induced Bid intracellular relocalization in murine L929 cells. Control L929-C2 and Hsp27-expressing L929-Hsp27 cells kept either untreated (NT) or exposed to staurosporine (1 μM) or etoposide (500 μM) for either 1, 2, or 4 h were lysed under conditions which preserve mitochondrial and membrane integrity as described in Materials and Methods. The resulting P20 and S cytosolic fractions were analyzed in immunoblots probed with anti-Bid antibody. Autoradiographs of immunoblots are shown.

    Article Snippet: Crystal violet, staurosporine, cytochalasin D, phalloidin, etoposide, and l -buthionine-( S , R )-sulfoximine (BSO) were purchased from Sigma.

    Techniques: Expressing, Western Blot

    Phalloidin counteracts the cytochalasin D-mediated release of cytochrome c from mitochondria and procaspase 3 activation. The effect is partial in case of staurosporine-treated cells. (A) Cytochrome c release analysis. Control (L929-C2) cells were either kept untreated or treated for 6 h with 0.5 μM cytochalasin D in the absence (−) or presence (+) of 2 μM phalloidin added to the culture medium 1 h before cytochalasin D. Cells were then processed for cytochrome c release analysis and proteins present in the different fractions were analyzed in immunoblots as described in Materials and Methods. The presence of cytochrome c (Cytc) and Hsc70 in the different fractions is shown. Autoradiographs of ECL-revealed immunoblots are presented. Note that phalloidin strongly decreases the release of cytochrome c induced by cytochalasin D. Lanes, P, pellet from untreated cells; lanes 0 and 6, soluble fractions isolated from untreated cells (0) or cells treated for 6 h with cytochalasin D. (B) Same as panel A, but in this case cells were treated with 1 μM staurosporine. (C) Caspase 3 activation in L929-C2 extracts isolated after 6 h of treatment with 0.5 μM cytochalasin D in the absence or presence of 2 μM phalloidin added to the culture medium 1 h before cytochalasin D. Activity of DEVD-specific caspases was then measured using the fluorescent substrate DEVD-AFC as described in Materials and Methods. (D) Same as panel C but in the presence of 1 μM staurosporine. (E) Same as panel C but in the presence of 500 μM etoposide. The activation index was determined as the ratio between the activity in extracts of treated cells to that measured in extracts of nontreated cells. The histogram shown is representative of three identical experiments; standard deviations (error bars) are presented ( n = 3). Note the protective activity of phalloidin.

    Journal: Molecular and Cellular Biology

    Article Title: Hsp27 as a Negative Regulator of Cytochrome c Release

    doi: 10.1128/MCB.22.3.816-834.2002

    Figure Lengend Snippet: Phalloidin counteracts the cytochalasin D-mediated release of cytochrome c from mitochondria and procaspase 3 activation. The effect is partial in case of staurosporine-treated cells. (A) Cytochrome c release analysis. Control (L929-C2) cells were either kept untreated or treated for 6 h with 0.5 μM cytochalasin D in the absence (−) or presence (+) of 2 μM phalloidin added to the culture medium 1 h before cytochalasin D. Cells were then processed for cytochrome c release analysis and proteins present in the different fractions were analyzed in immunoblots as described in Materials and Methods. The presence of cytochrome c (Cytc) and Hsc70 in the different fractions is shown. Autoradiographs of ECL-revealed immunoblots are presented. Note that phalloidin strongly decreases the release of cytochrome c induced by cytochalasin D. Lanes, P, pellet from untreated cells; lanes 0 and 6, soluble fractions isolated from untreated cells (0) or cells treated for 6 h with cytochalasin D. (B) Same as panel A, but in this case cells were treated with 1 μM staurosporine. (C) Caspase 3 activation in L929-C2 extracts isolated after 6 h of treatment with 0.5 μM cytochalasin D in the absence or presence of 2 μM phalloidin added to the culture medium 1 h before cytochalasin D. Activity of DEVD-specific caspases was then measured using the fluorescent substrate DEVD-AFC as described in Materials and Methods. (D) Same as panel C but in the presence of 1 μM staurosporine. (E) Same as panel C but in the presence of 500 μM etoposide. The activation index was determined as the ratio between the activity in extracts of treated cells to that measured in extracts of nontreated cells. The histogram shown is representative of three identical experiments; standard deviations (error bars) are presented ( n = 3). Note the protective activity of phalloidin.

    Article Snippet: Crystal violet, staurosporine, cytochalasin D, phalloidin, etoposide, and l -buthionine-( S , R )-sulfoximine (BSO) were purchased from Sigma.

    Techniques: Activation Assay, Western Blot, Isolation, Activity Assay

    Hsp27 expression interferes with cytochalasin D- and staurosporine-induced damages to F-actin. Fluorescence photomicrographs demonstrating the effect of cytochalasin D and staurosporine on F-actin fibers. Control L929-C2 cells (A to D) and human Hsp27-expressing L929-Hsp27 cells (E to H) were plated on glass plates and allowed to enter exponential cell growth phase for 24 h. They were then either kept untreated (A and E) or treated with 0.5 μM cytochalasin D (B and F), 1 μM staurosporine (C and G), or 500 μM etoposide (D and H). After 2 h of treatment the cells were fixed, stained with FITC-labeled phalloidin, and examined under a fluorescence microscope. The photomicrograph in panel G is enlarged in order to better detect the F-actin fibers (arrows), which are still visible in L929-Hsp27 cells treated with staurosporine. Bar, 10 μM.

    Journal: Molecular and Cellular Biology

    Article Title: Hsp27 as a Negative Regulator of Cytochrome c Release

    doi: 10.1128/MCB.22.3.816-834.2002

    Figure Lengend Snippet: Hsp27 expression interferes with cytochalasin D- and staurosporine-induced damages to F-actin. Fluorescence photomicrographs demonstrating the effect of cytochalasin D and staurosporine on F-actin fibers. Control L929-C2 cells (A to D) and human Hsp27-expressing L929-Hsp27 cells (E to H) were plated on glass plates and allowed to enter exponential cell growth phase for 24 h. They were then either kept untreated (A and E) or treated with 0.5 μM cytochalasin D (B and F), 1 μM staurosporine (C and G), or 500 μM etoposide (D and H). After 2 h of treatment the cells were fixed, stained with FITC-labeled phalloidin, and examined under a fluorescence microscope. The photomicrograph in panel G is enlarged in order to better detect the F-actin fibers (arrows), which are still visible in L929-Hsp27 cells treated with staurosporine. Bar, 10 μM.

    Article Snippet: Crystal violet, staurosporine, cytochalasin D, phalloidin, etoposide, and l -buthionine-( S , R )-sulfoximine (BSO) were purchased from Sigma.

    Techniques: Expressing, Fluorescence, Staining, Labeling, Microscopy

    Actin polymerization increases in response to DNA damage. A. U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, and images were captured at the indicated time points. B. The cell length and width were analyzed with Image J software in ≥100 cells per condition. C . U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, the intensity of phalloidin was measured with Image Pro Plus software. Scale bar, 10 µm. D. U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, and fluorescence assays were performed with a fluorescence microplate reader to measure cellular F-actin levels (phalloidin intensity/DAPI intensity). E. Cells were treated with ETO (10 µM) at indicated time points, and then whole cell extracts were analyzed by western blotting using anti-γH2AX antibody (a). U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h. Then, immunofluorescence was performed to detect the signal of γH2AX (b). Scale bar, 10 µm. All Statistical differences were determined by One-way ANOVA. Results are presented as means ± SD of values from three independent experiments. ETO, etoposide.

    Journal: PLoS ONE

    Article Title: Actin Polymerization Negatively Regulates p53 Function by Impairing Its Nuclear Import in Response to DNA Damage

    doi: 10.1371/journal.pone.0060179

    Figure Lengend Snippet: Actin polymerization increases in response to DNA damage. A. U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, and images were captured at the indicated time points. B. The cell length and width were analyzed with Image J software in ≥100 cells per condition. C . U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, the intensity of phalloidin was measured with Image Pro Plus software. Scale bar, 10 µm. D. U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h, and fluorescence assays were performed with a fluorescence microplate reader to measure cellular F-actin levels (phalloidin intensity/DAPI intensity). E. Cells were treated with ETO (10 µM) at indicated time points, and then whole cell extracts were analyzed by western blotting using anti-γH2AX antibody (a). U2OS cells were treated with ETO (10 µM) or untreated as control for 24 h. Then, immunofluorescence was performed to detect the signal of γH2AX (b). Scale bar, 10 µm. All Statistical differences were determined by One-way ANOVA. Results are presented as means ± SD of values from three independent experiments. ETO, etoposide.

    Article Snippet: Etoposide (ETO), a DNA damage inducer used in the present study, was from Sigma.

    Techniques: Software, Fluorescence, Western Blot, Immunofluorescence

    Validation of expression alterations in KIF20A in parallel with CDKN1A. Quantitative RT-PCR for analysis of gene expression alterations induced in TK6 cells after treatment for 4 h with three groups of compounds: (i) DNA damage-inducing genotoxins (clastogens); (ii) compounds that do not induce chromosomal aberrations; and (iii) genotoxins that do not involve DNA damage. Compound group (i) includes ENU, N -ethyl- N -nitrosourea; 5-FU, 5-fluorouracil; HU, hydroxyurea; ETOP, etoposide; EMS, ethyl methanesulfonate; MMS, methyl methanesulfonate; MMC, mitomycin C; CAMP, camptothecin; CP, cisplatin; CCNU, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea; and a newly-synthesized drug candidate (designated A) positive in the in vitro chromosomal aberration test. Compound group (ii) includes two newly-synthesized drug candidates (designated B and C) negative in the in vitro chromosomal aberration test. Compound group (iii) includes CHX, cycloheximide; COLCE, colcemid; COLCH, colchicines; NaCl, sodium chloride; ADE, adenine; TXL, paclitaxel; and 2-DA, 2-deoxyadenosine. The means ± standard deviation (SD) of cycle threshold ( C t) values were shown (single experiment in duplicate).

    Journal: International Journal of Molecular Sciences

    Article Title: Combinatorial Measurement of CDKN1A/p21 and KIF20A Expression for Discrimination of DNA Damage-Induced Clastogenicity

    doi: 10.3390/ijms151017256

    Figure Lengend Snippet: Validation of expression alterations in KIF20A in parallel with CDKN1A. Quantitative RT-PCR for analysis of gene expression alterations induced in TK6 cells after treatment for 4 h with three groups of compounds: (i) DNA damage-inducing genotoxins (clastogens); (ii) compounds that do not induce chromosomal aberrations; and (iii) genotoxins that do not involve DNA damage. Compound group (i) includes ENU, N -ethyl- N -nitrosourea; 5-FU, 5-fluorouracil; HU, hydroxyurea; ETOP, etoposide; EMS, ethyl methanesulfonate; MMS, methyl methanesulfonate; MMC, mitomycin C; CAMP, camptothecin; CP, cisplatin; CCNU, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea; and a newly-synthesized drug candidate (designated A) positive in the in vitro chromosomal aberration test. Compound group (ii) includes two newly-synthesized drug candidates (designated B and C) negative in the in vitro chromosomal aberration test. Compound group (iii) includes CHX, cycloheximide; COLCE, colcemid; COLCH, colchicines; NaCl, sodium chloride; ADE, adenine; TXL, paclitaxel; and 2-DA, 2-deoxyadenosine. The means ± standard deviation (SD) of cycle threshold ( C t) values were shown (single experiment in duplicate).

    Article Snippet: MMS, EMS, ETOP, HU, COLCH, ADE, ENU, CCNU, 5-FU, CAMP, TXL, 2-DA, and CHX were purchased from Sigma Aldrich.

    Techniques: Expressing, Quantitative RT-PCR, Synthesized, In Vitro, Standard Deviation

    Expression profiles of four genes on treatment with the genotoxins used in microarray analysis. The genes ATXN1 , KIF20A , KLF6 , and HBP1 , which are a part of CDKN1A -centered network, were downregulated in response to DNA damage. The log base 2 values of the ratio of alterations in the expression of these four downregulated genes to the mean values of the corresponding control obtained from the microarray data (single experiment in duplicate) are shown. TK6 cells were treated with MMC, mitomycin C; CP, cisplatin; MMS, methyl methanesulfonate; EMS, ethyl methanesulfonate; ETOP, etoposide; HU, hydroxyurea; COLCH, colchicine; and ADE, adenine; for 4 h at concentrations corresponding to 50% of relative cell growth (RCG) compared to number of cells in vehicle control, and allowed to recover for 20 h in standard cell culture medium.

    Journal: International Journal of Molecular Sciences

    Article Title: Combinatorial Measurement of CDKN1A/p21 and KIF20A Expression for Discrimination of DNA Damage-Induced Clastogenicity

    doi: 10.3390/ijms151017256

    Figure Lengend Snippet: Expression profiles of four genes on treatment with the genotoxins used in microarray analysis. The genes ATXN1 , KIF20A , KLF6 , and HBP1 , which are a part of CDKN1A -centered network, were downregulated in response to DNA damage. The log base 2 values of the ratio of alterations in the expression of these four downregulated genes to the mean values of the corresponding control obtained from the microarray data (single experiment in duplicate) are shown. TK6 cells were treated with MMC, mitomycin C; CP, cisplatin; MMS, methyl methanesulfonate; EMS, ethyl methanesulfonate; ETOP, etoposide; HU, hydroxyurea; COLCH, colchicine; and ADE, adenine; for 4 h at concentrations corresponding to 50% of relative cell growth (RCG) compared to number of cells in vehicle control, and allowed to recover for 20 h in standard cell culture medium.

    Article Snippet: MMS, EMS, ETOP, HU, COLCH, ADE, ENU, CCNU, 5-FU, CAMP, TXL, 2-DA, and CHX were purchased from Sigma Aldrich.

    Techniques: Expressing, Microarray, Cell Culture

    Etoposide treatment impairs the mRNA-binding potential of THOC5. ( A ) Two-sister culture of 4 × 10 6 MEF cells were treated with DMSO alone (DMSO) or with 10 μM of etoposide (etoposide) for 2 h. The cells were then extracted with lysis buffer.

    Journal: RNA

    Article Title: An ataxia-telangiectasia-mutated (ATM) kinase mediated response to DNA damage down-regulates the mRNA-binding potential of THOC5

    doi: 10.1261/rna.2820911

    Figure Lengend Snippet: Etoposide treatment impairs the mRNA-binding potential of THOC5. ( A ) Two-sister culture of 4 × 10 6 MEF cells were treated with DMSO alone (DMSO) or with 10 μM of etoposide (etoposide) for 2 h. The cells were then extracted with lysis buffer.

    Article Snippet: Two-sister culture of 4 × 106 MEF cells were treated with DMSO alone or with etoposide for 2 h. After washing three times, cells were lysed with lysis buffer (10 mM Tris, 150 mM NaCl, 1 mM PMSF, 0.5% NP40, protease inhibitor cocktail, [Sigma-Aldrich] and RNase inhibitor), and were then frozen and thawed three times.

    Techniques: Binding Assay, Lysis

    DNA damage drastically decreased the cytoplasmic pool of a set of THOC5-dependent mRNAs. ( A ) MEF cells were treated with DMSO alone (DMSO) or with 10 μM of etoposide (etoposide) for 2 h, and cell extracts were then subjected to THOC5, THOC1, THOC7,

    Journal: RNA

    Article Title: An ataxia-telangiectasia-mutated (ATM) kinase mediated response to DNA damage down-regulates the mRNA-binding potential of THOC5

    doi: 10.1261/rna.2820911

    Figure Lengend Snippet: DNA damage drastically decreased the cytoplasmic pool of a set of THOC5-dependent mRNAs. ( A ) MEF cells were treated with DMSO alone (DMSO) or with 10 μM of etoposide (etoposide) for 2 h, and cell extracts were then subjected to THOC5, THOC1, THOC7,

    Article Snippet: Two-sister culture of 4 × 106 MEF cells were treated with DMSO alone or with etoposide for 2 h. After washing three times, cells were lysed with lysis buffer (10 mM Tris, 150 mM NaCl, 1 mM PMSF, 0.5% NP40, protease inhibitor cocktail, [Sigma-Aldrich] and RNase inhibitor), and were then frozen and thawed three times.

    Techniques:

    Analysis of illegitimate genomic integration of the donor plasmid in ZFN treated cells . ( A ) Effect of etoposide treatment on the levels of illegitimate integration of the PuroR expression cassette from the pDonor in the genome of 293-Flp-mEGFP cells. After transfection with pDonor, cells were either left untreated or treated with etoposide before selection with puromycin in order to quantify the levels of illegitimate integration. As a control for the selection conditions, non-transfected cells were also included. Each bar represents the mean rate of puromycin resistant (R) cells ± SD from three parallels. ( B ) Quantification of illegitimate integration in 293-Flp-mEGFP cells after transfection with pDonor along with pZFN-L and pZFN-R. Controls included non-transfected cells and cells transfected with pZFNs or pDonor individually as indicated below the bars. The rates of illegitimate integration were normalized according to variations in PE. Statistical P values (paired t-test) comparing the means of the indicated bars are shown and an asterisk marks significant difference (P

    Journal: BMC Molecular Biology

    Article Title: Analysis of illegitimate genomic integration mediated by zinc-finger nucleases: implications for specificity of targeted gene correction

    doi: 10.1186/1471-2199-11-35

    Figure Lengend Snippet: Analysis of illegitimate genomic integration of the donor plasmid in ZFN treated cells . ( A ) Effect of etoposide treatment on the levels of illegitimate integration of the PuroR expression cassette from the pDonor in the genome of 293-Flp-mEGFP cells. After transfection with pDonor, cells were either left untreated or treated with etoposide before selection with puromycin in order to quantify the levels of illegitimate integration. As a control for the selection conditions, non-transfected cells were also included. Each bar represents the mean rate of puromycin resistant (R) cells ± SD from three parallels. ( B ) Quantification of illegitimate integration in 293-Flp-mEGFP cells after transfection with pDonor along with pZFN-L and pZFN-R. Controls included non-transfected cells and cells transfected with pZFNs or pDonor individually as indicated below the bars. The rates of illegitimate integration were normalized according to variations in PE. Statistical P values (paired t-test) comparing the means of the indicated bars are shown and an asterisk marks significant difference (P

    Article Snippet: Etoposide treatment of cells One day after transfection of 293-Flp-mEGFP cells with pDonor, cells were incubated overnight with growth medium containing 0.1 μM etoposide (Sigma-Aldrich).

    Techniques: Plasmid Preparation, Expressing, Transfection, Selection

    Silencing of SNAI2 phenocopies the effects of miR-203 re-expression on sensitization of U251AR cells to anticancer drugs and reversion of EMT (A) a. U251AR/shNC cells; b. U251AR/shSNAI2 cells. Light microscopy, 100× (a, b); Fluorescent microscopy, 100× (a, b). shSNAI2 and negative vector (shNC) were transfected into U251AR cells. At 48 h after transfection, fluorescent microscopy showed emission green fluorescence. (B) qRT-PCR validate the downregulation of SNAI2 after shRNA knockdown in U251AR cells. (C) Immunofluorescence analysis of the endogenous SNAI2 protein (red, left panels) in U251AR cells transfected with shSNAI2 or negative vector. Nuclei are stained in blue with DAPI. Scale bar, 20 μm. (D) The sensitivities of U251AR and U251AR/shSNAI2 to different concentrations of TMZ, imatinib and VP-16. (E) Morphology of U251AR cells transfected with negative vector or shSNAI2 vector. Scale bar, 100 μm. (F) SNAI2 knockdown reduces the invasion capacity of U251AR cells. Scale bar, 200 μm. (G) U251AR cell monolayer was transfected as indicated and scratched, then the migration of the cells towards the wound was visualised. Images were taken at various time points and Image J was used to determine the migration distance. (H) Western blotting show that silencing of SNAI2 can modulate the expression of EMT markers. VP-16, etoposide; TMZ, temozolomide. Data are presented as mean±s.d. of three independent experiments. * P

    Journal: Oncotarget

    Article Title: MiR-203 downregulation is responsible for chemoresistance in human glioblastoma by promoting epithelial-mesenchymal transition via SNAI2

    doi:

    Figure Lengend Snippet: Silencing of SNAI2 phenocopies the effects of miR-203 re-expression on sensitization of U251AR cells to anticancer drugs and reversion of EMT (A) a. U251AR/shNC cells; b. U251AR/shSNAI2 cells. Light microscopy, 100× (a, b); Fluorescent microscopy, 100× (a, b). shSNAI2 and negative vector (shNC) were transfected into U251AR cells. At 48 h after transfection, fluorescent microscopy showed emission green fluorescence. (B) qRT-PCR validate the downregulation of SNAI2 after shRNA knockdown in U251AR cells. (C) Immunofluorescence analysis of the endogenous SNAI2 protein (red, left panels) in U251AR cells transfected with shSNAI2 or negative vector. Nuclei are stained in blue with DAPI. Scale bar, 20 μm. (D) The sensitivities of U251AR and U251AR/shSNAI2 to different concentrations of TMZ, imatinib and VP-16. (E) Morphology of U251AR cells transfected with negative vector or shSNAI2 vector. Scale bar, 100 μm. (F) SNAI2 knockdown reduces the invasion capacity of U251AR cells. Scale bar, 200 μm. (G) U251AR cell monolayer was transfected as indicated and scratched, then the migration of the cells towards the wound was visualised. Images were taken at various time points and Image J was used to determine the migration distance. (H) Western blotting show that silencing of SNAI2 can modulate the expression of EMT markers. VP-16, etoposide; TMZ, temozolomide. Data are presented as mean±s.d. of three independent experiments. * P

    Article Snippet: After 24 h, the cells were treated with different concentrations of imatinib (Novartis, Basel, Switzerland), etoposide (VP-16) (Sigma Chemical Co., St. Louis, MO) and temozolomide (TMZ) (Sigma Chemical Co., St. Louis, MO), each at four concentrations ranging from 50 to 200 μg/ml for 48 h. The range of drug concentrations were based on earlier studies and aimed at obtaining IC50 values both for highly sensitive and resistant cases.

    Techniques: Expressing, Light Microscopy, Microscopy, Plasmid Preparation, Transfection, Fluorescence, Quantitative RT-PCR, shRNA, Immunofluorescence, Staining, Migration, Western Blot

    Re-expression of miR-203 in U251AR and U87AR cells sensitizes cells to anticancer drugs and reverses EMT while knockdown of miR-203 promotes resistance to anticancer drugs in U251 and U87 cells (A) qRT-PCR data validation of the downregulation of miR-203 in imatinib-resistant GBM cells compared with their parental cells, normalized to U6RNA, which was obtained from miRNA microarrays. (B, C) The sensitivities of U251AR and U87AR cells to imatinib, VP-16 and TMZ after transfected with miR-203 or miRNAs control. (D, E) Transfection with anti-miR-203 promotes resistance to imatinib, VP-16 and TMZ in U251 and U87 cells. (F) Morphology of U251AR and U87AR cells transfected with miRNA control or miR-203. Scale bar, 100 μm. (G) Western blotting show that re-expression of miR-203 modulates the expression of EMT markers. (H, I) U251AR and U87AR cells were transfected with miR-203 or anti-miR-203, and then collected for transwell invasion assay or wound healing assay. Shown were pictures of representative fields for each experiment. Scale bar, 200 μm. Data were expressed as mean±s.d. from three independent experiments. VP-16, etoposide; TMZ, temozolomide. * P

    Journal: Oncotarget

    Article Title: MiR-203 downregulation is responsible for chemoresistance in human glioblastoma by promoting epithelial-mesenchymal transition via SNAI2

    doi:

    Figure Lengend Snippet: Re-expression of miR-203 in U251AR and U87AR cells sensitizes cells to anticancer drugs and reverses EMT while knockdown of miR-203 promotes resistance to anticancer drugs in U251 and U87 cells (A) qRT-PCR data validation of the downregulation of miR-203 in imatinib-resistant GBM cells compared with their parental cells, normalized to U6RNA, which was obtained from miRNA microarrays. (B, C) The sensitivities of U251AR and U87AR cells to imatinib, VP-16 and TMZ after transfected with miR-203 or miRNAs control. (D, E) Transfection with anti-miR-203 promotes resistance to imatinib, VP-16 and TMZ in U251 and U87 cells. (F) Morphology of U251AR and U87AR cells transfected with miRNA control or miR-203. Scale bar, 100 μm. (G) Western blotting show that re-expression of miR-203 modulates the expression of EMT markers. (H, I) U251AR and U87AR cells were transfected with miR-203 or anti-miR-203, and then collected for transwell invasion assay or wound healing assay. Shown were pictures of representative fields for each experiment. Scale bar, 200 μm. Data were expressed as mean±s.d. from three independent experiments. VP-16, etoposide; TMZ, temozolomide. * P

    Article Snippet: After 24 h, the cells were treated with different concentrations of imatinib (Novartis, Basel, Switzerland), etoposide (VP-16) (Sigma Chemical Co., St. Louis, MO) and temozolomide (TMZ) (Sigma Chemical Co., St. Louis, MO), each at four concentrations ranging from 50 to 200 μg/ml for 48 h. The range of drug concentrations were based on earlier studies and aimed at obtaining IC50 values both for highly sensitive and resistant cases.

    Techniques: Expressing, Quantitative RT-PCR, Transfection, Western Blot, Transwell Invasion Assay, Wound Healing Assay

    SNAI2 contributes to chemoresistance and EMT in GBM cells (A) Overexpression of SNAI2 promotes resistance to imatinib, VP-16 and TMZ. (B) Morphology of U87 cells transfected with pcDNA3.1-mock or pcDNA3.1-SNAI2. Scale bar, 100 μm. (C) Invasion of U87 cells after pcDNA3.1-SNAI2 transfection. Scale bar, 200 μm. (D) Protein expression of EMT markers in U87 cells transfected with pcDNA3.1-mock or pcDNA3.1-SNAI2, determined by western blotting. (E) The sensitivities to imatinib, VP-16 and TMZ were measured after cells transfected with indicated constructs and miR-203 in U251AR. (F) Invasion assay of U251AR cells expressing indicated vectors and miR-203. (G) qRT-PCR for EMT markers in U251AR cells expressing indicated constructs and miR-203. * P

    Journal: Oncotarget

    Article Title: MiR-203 downregulation is responsible for chemoresistance in human glioblastoma by promoting epithelial-mesenchymal transition via SNAI2

    doi:

    Figure Lengend Snippet: SNAI2 contributes to chemoresistance and EMT in GBM cells (A) Overexpression of SNAI2 promotes resistance to imatinib, VP-16 and TMZ. (B) Morphology of U87 cells transfected with pcDNA3.1-mock or pcDNA3.1-SNAI2. Scale bar, 100 μm. (C) Invasion of U87 cells after pcDNA3.1-SNAI2 transfection. Scale bar, 200 μm. (D) Protein expression of EMT markers in U87 cells transfected with pcDNA3.1-mock or pcDNA3.1-SNAI2, determined by western blotting. (E) The sensitivities to imatinib, VP-16 and TMZ were measured after cells transfected with indicated constructs and miR-203 in U251AR. (F) Invasion assay of U251AR cells expressing indicated vectors and miR-203. (G) qRT-PCR for EMT markers in U251AR cells expressing indicated constructs and miR-203. * P

    Article Snippet: After 24 h, the cells were treated with different concentrations of imatinib (Novartis, Basel, Switzerland), etoposide (VP-16) (Sigma Chemical Co., St. Louis, MO) and temozolomide (TMZ) (Sigma Chemical Co., St. Louis, MO), each at four concentrations ranging from 50 to 200 μg/ml for 48 h. The range of drug concentrations were based on earlier studies and aimed at obtaining IC50 values both for highly sensitive and resistant cases.

    Techniques: Over Expression, Transfection, Expressing, Western Blot, Construct, Invasion Assay, Quantitative RT-PCR

    DBC1 did not affect SIRT1 activity in vitro in liver cancer cells. A549 and SNU-182 (A and B) cells were transfected with siRNAs targeting SIRT1 or DBC1 (50 or 200 nM). Etoposide (20 μ M) was administered for 12 h to induce p53 hyperacetylation due to DNA damage. Knockdown of SIRT1 or DBC1 and acetylation of p53 were evaluated by western blotting with the indicated antibodies. All membranes were probed for GAPDH to confirm equal protein loading. SIRT1, silent mating type information regulation 2 homolog 1; DBC1, deleted in breast cancer-1.

    Journal: Oncology Letters

    Article Title: DBC1 does not function as a negative regulator of SIRT1 in liver cancer

    doi: 10.3892/ol.2012.875

    Figure Lengend Snippet: DBC1 did not affect SIRT1 activity in vitro in liver cancer cells. A549 and SNU-182 (A and B) cells were transfected with siRNAs targeting SIRT1 or DBC1 (50 or 200 nM). Etoposide (20 μ M) was administered for 12 h to induce p53 hyperacetylation due to DNA damage. Knockdown of SIRT1 or DBC1 and acetylation of p53 were evaluated by western blotting with the indicated antibodies. All membranes were probed for GAPDH to confirm equal protein loading. SIRT1, silent mating type information regulation 2 homolog 1; DBC1, deleted in breast cancer-1.

    Article Snippet: To induce DNA damage, cells were treated with 20 μ M etoposide (Sigma) for 12 h.

    Techniques: Activity Assay, In Vitro, Transfection, Western Blot

    Targeted disruptions of DBC1 or SIRT1 reduced liver cancer cell viability. HepG2 (A) and SNU-182 (B) cells were transfected with siRNAs targeting SIRT1 or DBC1 (50 or 200 nM), and then treated with 20 μ M of etoposide for 12 h. Cell viabilities were determined by MTT assay as described in Materials and methods. Data are presented as means ± standard deviation of three experiments (unpaired Student’s t-test, * P

    Journal: Oncology Letters

    Article Title: DBC1 does not function as a negative regulator of SIRT1 in liver cancer

    doi: 10.3892/ol.2012.875

    Figure Lengend Snippet: Targeted disruptions of DBC1 or SIRT1 reduced liver cancer cell viability. HepG2 (A) and SNU-182 (B) cells were transfected with siRNAs targeting SIRT1 or DBC1 (50 or 200 nM), and then treated with 20 μ M of etoposide for 12 h. Cell viabilities were determined by MTT assay as described in Materials and methods. Data are presented as means ± standard deviation of three experiments (unpaired Student’s t-test, * P

    Article Snippet: To induce DNA damage, cells were treated with 20 μ M etoposide (Sigma) for 12 h.

    Techniques: Transfection, MTT Assay, Standard Deviation

    Trib2 overexpressing cells are chemotherapy resistant Cell viability of (A) NB4 (B) HL60 (C) U937 overexpressing Trib2 cells, in response to increasing doses of cytarabine (Ara-C), Doxocyline (Dox), daunorubicin (DNR) and Etoposide (Etop) after 24hr, determined by Trypan blue exclusion. (D) Representative apoptosis profile of DNR treated U937 cells transduced with Trib2 or PHR empty vector for 24hr; live, DAPI−Annexin V−; early apoptotic, DAPI− Annexin V+; late apoptotic, DAPI+ Annexin V+. (E) Graph of the average fold change in cells positive for Annexin V, in U937 cells transduced with Trib2 or PHR empty vector and treated with DNR for 24hr, from 3 independent experiments. Error bars denote +/− SEM. (F) Representative mitosis FACS profile of DNR treated U937 cells transduced with Trib2 or PHR empty vector for 24hrs. (G) Graph of fold change in mitotic index (MI), following treatment with DNR for 24hr and normalized to untreated control sample. Error bars denote +/− SEM. (H) U937 cells were transduced with control pLKO.1 or pLKO.1 shTrib2 lentivirus and treated with cytarabine (Ara-C), daunorubicin (DNR) and etoposide (Etop). Fold change in cell death (Annexin+ and/or DAPI+ cells) was determined after 24hrs. Bars represent the average of 2 independent experiments each with 3 technical replicates. Error bars denote +/− SEM.

    Journal: Oncotarget

    Article Title: Trib2 expression in granulocyte-monocyte progenitors drives a highly drug resistant acute myeloid leukaemia linked to elevated Bcl2

    doi: 10.18632/oncotarget.24525

    Figure Lengend Snippet: Trib2 overexpressing cells are chemotherapy resistant Cell viability of (A) NB4 (B) HL60 (C) U937 overexpressing Trib2 cells, in response to increasing doses of cytarabine (Ara-C), Doxocyline (Dox), daunorubicin (DNR) and Etoposide (Etop) after 24hr, determined by Trypan blue exclusion. (D) Representative apoptosis profile of DNR treated U937 cells transduced with Trib2 or PHR empty vector for 24hr; live, DAPI−Annexin V−; early apoptotic, DAPI− Annexin V+; late apoptotic, DAPI+ Annexin V+. (E) Graph of the average fold change in cells positive for Annexin V, in U937 cells transduced with Trib2 or PHR empty vector and treated with DNR for 24hr, from 3 independent experiments. Error bars denote +/− SEM. (F) Representative mitosis FACS profile of DNR treated U937 cells transduced with Trib2 or PHR empty vector for 24hrs. (G) Graph of fold change in mitotic index (MI), following treatment with DNR for 24hr and normalized to untreated control sample. Error bars denote +/− SEM. (H) U937 cells were transduced with control pLKO.1 or pLKO.1 shTrib2 lentivirus and treated with cytarabine (Ara-C), daunorubicin (DNR) and etoposide (Etop). Fold change in cell death (Annexin+ and/or DAPI+ cells) was determined after 24hrs. Bars represent the average of 2 independent experiments each with 3 technical replicates. Error bars denote +/− SEM.

    Article Snippet: The cells were treated as indicated with cytarabine (Ara-C), Etoposide (Etop), doxorubicin (DOX), or daunorubicin (DNR) (Sigma-Aldrich), at the concentration indicated.

    Techniques: Acetylene Reduction Assay, Transduction, Plasmid Preparation, FACS

    miR-3196 inhibits VP-16 induced apoptosis by downregulating PUMA A. A549 cells were transfected with miR-3196 mimics (miR-3196) and/or combined with pcDNA3-PUMA (PUMA) for 24 h, followed by treatment with VP16 (100 μM) for 48 h. The apoptotic cells were detected by FCM (left panel). Error bars denote the mean ± SD (right panel). B. H1650 cells were transfected and treated as in (A). The apoptotic cells were detected by FCM (left panel). Error bars denote the mean ± SD (right panel). * P

    Journal: Oncotarget

    Article Title: MicroRNA-3196 is inhibited by H2AX phosphorylation and attenuates lung cancer cell apoptosis by downregulating PUMA

    doi: 10.18632/oncotarget.12794

    Figure Lengend Snippet: miR-3196 inhibits VP-16 induced apoptosis by downregulating PUMA A. A549 cells were transfected with miR-3196 mimics (miR-3196) and/or combined with pcDNA3-PUMA (PUMA) for 24 h, followed by treatment with VP16 (100 μM) for 48 h. The apoptotic cells were detected by FCM (left panel). Error bars denote the mean ± SD (right panel). B. H1650 cells were transfected and treated as in (A). The apoptotic cells were detected by FCM (left panel). Error bars denote the mean ± SD (right panel). * P

    Article Snippet: Cells were grown to 80% confluence and then serum-deprived for 12 h prior to etoposide (VP16) (Sigma–Aldrich, St Louis, MO, USA) stimulation.

    Techniques: Transfection

    Quantification of apoptosis in PAX3 siRNA transfected SH-SY5Y (A and B) and SH-EP1 (C and D) cells following the treatment with chemotherapeutic drugs. (A) Depicts representative immunofluorescence staining of control siRNA and PAX3 siRNA#4 transfected SH-SY5Y cells which were exposed to drugs at IC 50 for 24 hrs for annexin V (green) and propidium iodide (red). (B) Histograms show apoptosis as measured by the percentage of cells with pre-G1 DNA content among control siRNA or PAX3 siRNA#4 transfected SH-SY5Y cells treated with etoposide, vincristine and cisplatin at IC 50 and IC 80 /IC 90 for 6, 24, 48 and 72 hrs. (C) Depicts representative immunofluorescence staining of control siRNA and PAX3 siRNA#4 transfected SH-EP1 cells which were exposed to drugs at IC 90 for 6 hrs for annexin V (green) and propidium iodide (red). (D) Histograms show apoptosis as measured by the percentage of cells with pre-G1 DNA content among control siRNA or PAX3 siRNA#4 transfected SH-EP1 cells treated with etoposide, vincristine and cisplatin at IC 50 and IC 90 for 6, 24, 48 and 72 hrs. Scale bars represent 20 μm. Error bars represent mean ± SD for two independent experiments. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: PAX3 in neuroblastoma: oncogenic potential, chemosensitivity and signalling pathways

    doi: 10.1111/jcmm.12155

    Figure Lengend Snippet: Quantification of apoptosis in PAX3 siRNA transfected SH-SY5Y (A and B) and SH-EP1 (C and D) cells following the treatment with chemotherapeutic drugs. (A) Depicts representative immunofluorescence staining of control siRNA and PAX3 siRNA#4 transfected SH-SY5Y cells which were exposed to drugs at IC 50 for 24 hrs for annexin V (green) and propidium iodide (red). (B) Histograms show apoptosis as measured by the percentage of cells with pre-G1 DNA content among control siRNA or PAX3 siRNA#4 transfected SH-SY5Y cells treated with etoposide, vincristine and cisplatin at IC 50 and IC 80 /IC 90 for 6, 24, 48 and 72 hrs. (C) Depicts representative immunofluorescence staining of control siRNA and PAX3 siRNA#4 transfected SH-EP1 cells which were exposed to drugs at IC 90 for 6 hrs for annexin V (green) and propidium iodide (red). (D) Histograms show apoptosis as measured by the percentage of cells with pre-G1 DNA content among control siRNA or PAX3 siRNA#4 transfected SH-EP1 cells treated with etoposide, vincristine and cisplatin at IC 50 and IC 90 for 6, 24, 48 and 72 hrs. Scale bars represent 20 μm. Error bars represent mean ± SD for two independent experiments. * P

    Article Snippet: Cytotoxicity induced by chemotherapeutic drugs Etoposide and cisplatin were dissolved in dimethyl sulphoxide (DMSO) and vincristine in methanol (all from Sigma-Aldrich), stored at −20°C, further diluted in growth medium immediately before use (the final concentrations of DMSO and methanol were < 0.04).

    Techniques: Transfection, Immunofluorescence, Staining

    HT1080 FUCCI show strong cell cycle-associated cell death. Cell cycle-associated death standards are used. ( a ) A representative FUCCI trace of cells treated with the topoisomerase-α poison, etoposide. Cells progress from G1-phase (red), with normal kinetics, progress to a green state and die, consistent with S/G2-phase associated death. ( b ) A representative FUCCI trace of cells treated with the DNA modifier, cisplatin. Cells most often progress normally from G1-phase (red) to an all green state and die, consistent with S-phase associated death. ( c ) A representative FUCCI trace of cells treated with a Kinesin-5 inhibitor, K5I. This cell progresses through the cell cycle with normal kinetics and enters mitotic arrest at 14 h post-treatment (*). While arrested, red signal is reacquired after 3–4 h, beginning at 17 h. This cell dies at 23 h and nearly all other cells also die while arrested in mitosis. Arrows indicate time of death. See Supplementary Fig. 1e–g online for FUCCI distributions over time. Supplementary video S6-8 online. Cell number tracked: etoposide, 33, cisplatin, 21, K5I, 30.

    Journal: Scientific Reports

    Article Title: Longitudinal tracking of single live cancer cells to understand cell cycle effects of the nuclear export inhibitor, selinexor

    doi: 10.1038/srep14391

    Figure Lengend Snippet: HT1080 FUCCI show strong cell cycle-associated cell death. Cell cycle-associated death standards are used. ( a ) A representative FUCCI trace of cells treated with the topoisomerase-α poison, etoposide. Cells progress from G1-phase (red), with normal kinetics, progress to a green state and die, consistent with S/G2-phase associated death. ( b ) A representative FUCCI trace of cells treated with the DNA modifier, cisplatin. Cells most often progress normally from G1-phase (red) to an all green state and die, consistent with S-phase associated death. ( c ) A representative FUCCI trace of cells treated with a Kinesin-5 inhibitor, K5I. This cell progresses through the cell cycle with normal kinetics and enters mitotic arrest at 14 h post-treatment (*). While arrested, red signal is reacquired after 3–4 h, beginning at 17 h. This cell dies at 23 h and nearly all other cells also die while arrested in mitosis. Arrows indicate time of death. See Supplementary Fig. 1e–g online for FUCCI distributions over time. Supplementary video S6-8 online. Cell number tracked: etoposide, 33, cisplatin, 21, K5I, 30.

    Article Snippet: PD0332991, Etoposide (VP-16), and cisplatin are from Selleckchem (Houston, TX); stock solutions re in DMSO except for cisplatin, which is in dimethylformamide.

    Techniques:

    Longitudinal single cell tracking with survival analysis reveals cell cycle-associated responses of selinexor. HT1080 FUCCI cells. ( a ) Percent survival after treatment with cell cycle drugs, selinexor and controls. 100% of cells have divided by ~16 h for untreated (black) and KPT 301 treated (blueberry) cells; dashed lines represent the daughter cell population. Half of selinexor-treated cells are lost by ~55 h (maraschino) and the rate of loss is most similar to the S-phase associated drug, etoposide (honeydew); cisplatin (grape) and K5I (avocado) are comparatively very potent killers. ( b ) Survival curve for selinexor treated cells separated by FUCCI status upon treatment. Cells treated in early S-phase (yellow) die the fastest. Cells treated in late S/G2-phase (green) show little death and instead divide (dashed green line). Cells treated in G1-phase (red) and daughter cells from treated late S/G2-phase cells die at very similar rates. ( c , d ) Two-axis and violin plots for all cells that die after selinexor treatment or that die after being born into selinexor. Two-axis plots show FUCCI status upon treatment on the left axis and upon death on the right. Violin plot shows timing of death and FUCCI status (red triangle for G1-phase, yellow square for early S-phase, green circle for S/G2-phase, and blue star for mitosis) upon death. For ( d ) the FUCCI status of parent cells upon treatment are on the left axis and FUCCI status of daughter cells upon death on the right –84% of cells that die after dividing in selinexor, die in G1-phase (~84%). ( e ) Continuously tracked cells to obtain fraction of time spent in each FUCCI stage for each condition and table indicating the average life-span of cells for each condition; selinexor treated cells live 42 h on average, and spend increased time in G1-phase in particular (see Table 1 ). Supplementary videos S15-18 online. Cell numbers scored: ( a – d ) untreated, 42, selinexor, 376, KPT 301, 47, etoposide, 84, cisplatin, 54, K5I, 51. ( e ) Cell number tracked: untreated, 22, PD0332991, 19, aphidicolin, 24, RO-3306, 20, etoposide, 33, cisplatin, 21, K5I, 30, KPT 301, 27, selinexor, 117.

    Journal: Scientific Reports

    Article Title: Longitudinal tracking of single live cancer cells to understand cell cycle effects of the nuclear export inhibitor, selinexor

    doi: 10.1038/srep14391

    Figure Lengend Snippet: Longitudinal single cell tracking with survival analysis reveals cell cycle-associated responses of selinexor. HT1080 FUCCI cells. ( a ) Percent survival after treatment with cell cycle drugs, selinexor and controls. 100% of cells have divided by ~16 h for untreated (black) and KPT 301 treated (blueberry) cells; dashed lines represent the daughter cell population. Half of selinexor-treated cells are lost by ~55 h (maraschino) and the rate of loss is most similar to the S-phase associated drug, etoposide (honeydew); cisplatin (grape) and K5I (avocado) are comparatively very potent killers. ( b ) Survival curve for selinexor treated cells separated by FUCCI status upon treatment. Cells treated in early S-phase (yellow) die the fastest. Cells treated in late S/G2-phase (green) show little death and instead divide (dashed green line). Cells treated in G1-phase (red) and daughter cells from treated late S/G2-phase cells die at very similar rates. ( c , d ) Two-axis and violin plots for all cells that die after selinexor treatment or that die after being born into selinexor. Two-axis plots show FUCCI status upon treatment on the left axis and upon death on the right. Violin plot shows timing of death and FUCCI status (red triangle for G1-phase, yellow square for early S-phase, green circle for S/G2-phase, and blue star for mitosis) upon death. For ( d ) the FUCCI status of parent cells upon treatment are on the left axis and FUCCI status of daughter cells upon death on the right –84% of cells that die after dividing in selinexor, die in G1-phase (~84%). ( e ) Continuously tracked cells to obtain fraction of time spent in each FUCCI stage for each condition and table indicating the average life-span of cells for each condition; selinexor treated cells live 42 h on average, and spend increased time in G1-phase in particular (see Table 1 ). Supplementary videos S15-18 online. Cell numbers scored: ( a – d ) untreated, 42, selinexor, 376, KPT 301, 47, etoposide, 84, cisplatin, 54, K5I, 51. ( e ) Cell number tracked: untreated, 22, PD0332991, 19, aphidicolin, 24, RO-3306, 20, etoposide, 33, cisplatin, 21, K5I, 30, KPT 301, 27, selinexor, 117.

    Article Snippet: PD0332991, Etoposide (VP-16), and cisplatin are from Selleckchem (Houston, TX); stock solutions re in DMSO except for cisplatin, which is in dimethylformamide.

    Techniques: Single Cell Tracking

    Etoposide and ANS-etoposide toxicity in GSTA1 and MGST1 overexpressing cells. Cellular toxicity of (A) etoposide and (B) ANS–etoposide analyzed with the MTT assay after short-term 24 h exposure with indicated doses, in at least three independent experiments measured in at least triplicate (mean ± SEM, n ≥ 9, * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001). Left panel: MGST1 overexpressing MCF7 cells (dark gray/MGST1) and the vector control (light gray/control). Right panel: GSTA1 overexpressing V79 cells (dark gray/GSTA1) and the vector control (light gray/control).

    Journal: Molecular pharmaceutics

    Article Title: Chemical Reactivity Window Determines Prodrug Efficiency toward Glutathione Transferase Overexpressing Cancer Cells

    doi: 10.1021/acs.molpharmaceut.6b00140

    Figure Lengend Snippet: Etoposide and ANS-etoposide toxicity in GSTA1 and MGST1 overexpressing cells. Cellular toxicity of (A) etoposide and (B) ANS–etoposide analyzed with the MTT assay after short-term 24 h exposure with indicated doses, in at least three independent experiments measured in at least triplicate (mean ± SEM, n ≥ 9, * P ≤ 0.05, ** P ≤ 0.01, and *** P ≤ 0.001). Left panel: MGST1 overexpressing MCF7 cells (dark gray/MGST1) and the vector control (light gray/control). Right panel: GSTA1 overexpressing V79 cells (dark gray/GSTA1) and the vector control (light gray/control).

    Article Snippet: 1-Chloro-2,4-dinitrobenzene (CDNB), reduced glutathione, and doxorubicin were purchased from Sigma-Aldrich, whereas etoposide was purchased from Vepesid, Bristol-Myers.

    Techniques: MTT Assay, Plasmid Preparation

    Chemical structures and conversion of prodrugs. (A) Chemical structures of DOX and etoposide as well as the prodrugs MNS–DOX, DNS-DOX, ANS–OX, and ANS–etoposide are depicted. (B) Conversion of the prodrug yields a free doxorubicin/etoposide, sulfur dioxide and an acetylmononitro/dinitro benzene moiety conjugated to GSH. These prodrugs are hypothesized to enter the cell via a passive diffusion. In the cell, GSTs are able to activate the prodrugs via their sulfonamidase and sulfonamide cleavage activity.

    Journal: Molecular pharmaceutics

    Article Title: Chemical Reactivity Window Determines Prodrug Efficiency toward Glutathione Transferase Overexpressing Cancer Cells

    doi: 10.1021/acs.molpharmaceut.6b00140

    Figure Lengend Snippet: Chemical structures and conversion of prodrugs. (A) Chemical structures of DOX and etoposide as well as the prodrugs MNS–DOX, DNS-DOX, ANS–OX, and ANS–etoposide are depicted. (B) Conversion of the prodrug yields a free doxorubicin/etoposide, sulfur dioxide and an acetylmononitro/dinitro benzene moiety conjugated to GSH. These prodrugs are hypothesized to enter the cell via a passive diffusion. In the cell, GSTs are able to activate the prodrugs via their sulfonamidase and sulfonamide cleavage activity.

    Article Snippet: 1-Chloro-2,4-dinitrobenzene (CDNB), reduced glutathione, and doxorubicin were purchased from Sigma-Aldrich, whereas etoposide was purchased from Vepesid, Bristol-Myers.

    Techniques: Diffusion-based Assay, Activity Assay

    Synthesis of ANS–DOX (1) and ANS–Etoposide (2); See Materials and Methods for Details

    Journal: Molecular pharmaceutics

    Article Title: Chemical Reactivity Window Determines Prodrug Efficiency toward Glutathione Transferase Overexpressing Cancer Cells

    doi: 10.1021/acs.molpharmaceut.6b00140

    Figure Lengend Snippet: Synthesis of ANS–DOX (1) and ANS–Etoposide (2); See Materials and Methods for Details

    Article Snippet: 1-Chloro-2,4-dinitrobenzene (CDNB), reduced glutathione, and doxorubicin were purchased from Sigma-Aldrich, whereas etoposide was purchased from Vepesid, Bristol-Myers.

    Techniques:

    HDAC4 is dysregulated during senescence and aging and is required for senescence escape. a. Immunoblot analysis in IMR90 cells undergoing replicative senescence. Actin was used as loading control. b. Microscopic images of SA-β-gal stained IMR90 cells (scale bar 50 µm). c. Immunoblot analysis in tissue-derived lysates obtained from C57BL/6J female mice sacrificed at 128 (young) and 774 (old) days of age. Actin was used as loading control. d. Immunoblot analysis in BJ/ hTERT expressing the indicated transgenes for the indicated time. Vimentin was the loading control. e. Cellular lysates obtained in BJ/ hTERT expressing for 8 days the indicated transgenes and treated or not for 8h with MG132 were immunoprecipitated using anti-HDAC4 and immunoblotted with the indicated antibodies. f. Immunoblot analysis in BJ/ hTERT cells expressing HRAS G12V and silenced for GSK3β, as indicated. g. Immunoblot analysis in BJ/ hTERT/E1A/RAS / HDAC4 + / + or -/- cells, as indicated. Actin was used as loading control. h. Analysis of the senescent cells as scored after SA-β-gal staining. Mean ± SD; n = 3. i. Immunoblot analysis in SK-LMS-1/ HDAC4 + / + or -/- as indicated. j. Cell-proliferation curve of the indicated HDAC4 + / +, + /-, -/- SK-LMS-1 cells. Mean ± SD; n = 4. k. Analysis of the senescent cells as scored after SA-β-gal staining. Mean ± SD; n = 4. l. Representative image of normal and altered DAPI-stained nuclei observed in SK-LMS-1 HDAC4 -/- cells (scale bar 10 µm). m-n. Analysis of the % of cells displaying altered nuclei or γH2AX foci ( > 5). Etoposide was a control (2h, 20µM). Mean ± SD; n = 4. o-p-q. Analysis of SA-β-gal (o), BrdU (p) and γH2AX (q) positivity in wt or HDAC4 KO cells expressing HYGRO R (clone 635) or HDAC4 PAM . Mean ± SD; n = 4. The significance is relative to clone 635. r. Immunoblot analysis in SK-LMS-1 wt or KO (clone 66) cells re-expressing a tamoxifen inducible HDAC4 PAM -ER . Arrowheads point to HDAC4 cleavage products observed in HDAC4-ER expressing cells. Actin was the loading control. s-v. Analysis of SA-β-gal (s), BrdU (t), nuclear alteration (u) and γH2AX (v) positivity in the indicated cells. Mean ± SD; n = 4.

    Journal: bioRxiv

    Article Title: HDAC4 controls senescence and aging by safeguarding the epigenetic identity and ensuring the genomic integrity

    doi: 10.1101/2020.06.04.132787

    Figure Lengend Snippet: HDAC4 is dysregulated during senescence and aging and is required for senescence escape. a. Immunoblot analysis in IMR90 cells undergoing replicative senescence. Actin was used as loading control. b. Microscopic images of SA-β-gal stained IMR90 cells (scale bar 50 µm). c. Immunoblot analysis in tissue-derived lysates obtained from C57BL/6J female mice sacrificed at 128 (young) and 774 (old) days of age. Actin was used as loading control. d. Immunoblot analysis in BJ/ hTERT expressing the indicated transgenes for the indicated time. Vimentin was the loading control. e. Cellular lysates obtained in BJ/ hTERT expressing for 8 days the indicated transgenes and treated or not for 8h with MG132 were immunoprecipitated using anti-HDAC4 and immunoblotted with the indicated antibodies. f. Immunoblot analysis in BJ/ hTERT cells expressing HRAS G12V and silenced for GSK3β, as indicated. g. Immunoblot analysis in BJ/ hTERT/E1A/RAS / HDAC4 + / + or -/- cells, as indicated. Actin was used as loading control. h. Analysis of the senescent cells as scored after SA-β-gal staining. Mean ± SD; n = 3. i. Immunoblot analysis in SK-LMS-1/ HDAC4 + / + or -/- as indicated. j. Cell-proliferation curve of the indicated HDAC4 + / +, + /-, -/- SK-LMS-1 cells. Mean ± SD; n = 4. k. Analysis of the senescent cells as scored after SA-β-gal staining. Mean ± SD; n = 4. l. Representative image of normal and altered DAPI-stained nuclei observed in SK-LMS-1 HDAC4 -/- cells (scale bar 10 µm). m-n. Analysis of the % of cells displaying altered nuclei or γH2AX foci ( > 5). Etoposide was a control (2h, 20µM). Mean ± SD; n = 4. o-p-q. Analysis of SA-β-gal (o), BrdU (p) and γH2AX (q) positivity in wt or HDAC4 KO cells expressing HYGRO R (clone 635) or HDAC4 PAM . Mean ± SD; n = 4. The significance is relative to clone 635. r. Immunoblot analysis in SK-LMS-1 wt or KO (clone 66) cells re-expressing a tamoxifen inducible HDAC4 PAM -ER . Arrowheads point to HDAC4 cleavage products observed in HDAC4-ER expressing cells. Actin was the loading control. s-v. Analysis of SA-β-gal (s), BrdU (t), nuclear alteration (u) and γH2AX (v) positivity in the indicated cells. Mean ± SD; n = 4.

    Article Snippet: The following chemicals were used: 250nM 4-OHT (Sigma-Aldrich), 1µM Doxycycline (Sigma-Aldrich), 1µM MG132 (Sigma-Aldrich), 10µM Chloroquine (Sigma-Aldrich), 0.4% Trypan Blue (Sigma-Aldrich), 200µM H2 O2 (Sigma-Aldrich), 1µM PD0332991 (Sigma-Aldrich), 500nM Aphidicolin (Sigma-Aldrich), 3.125µm Camptothecin (Enzo Life Sciences), 20µM Etoposide (Enzo Life Sciences), 100nM ABT-263 (Clinisciences).

    Techniques: Staining, Derivative Assay, Mouse Assay, Expressing, Immunoprecipitation

    A viability metric reports overall dose response and heterogeneity based on relative esterase activity and membrane permeability. In (a), representative thumbnails from merged images of esterase-cleaved calcein AM (green) and intercalated ethidium bromide (red) in ovarian cancer 3D overlays for (left to right), no treatment (NT) and total killing (TK) and increasing doses of paclitaxel (as a representative cytotoxic agent). Thumbnails qualitatively display increasing ethidium bromide intercalation (red) and decreasing esterase activity (green) with increasing cytotoxic dose similar for all therapies assayed here. Scale bars = 500 μm. In (b), mean fluorescence signals from full fields after rescaling ( Equation 1 ) are shown for escalating doses of paclitaxel, carboplatin, etoposide, and verteporfin PDT treatments (from left to right) in the upper plots used to quantify overall viability (lower plots, from Equation 2 ) and estimate fractional lethal doses (LD). In (c), distributions of nodule viabilities ( N > 1000 ) from automated segmentation (shown for paclitaxel-treated cultures) shifts to the left as expected with increasing dose. In (d), paclitaxel and carboplatin viability distributions at LD80 (thumbnails, left, scalebars = 500 μm) are juxtaposed to reveal a relatively tight distribution in the former in contrast to heterogeneous viability spectrum in the latter with sub-populations up to 80–90% viable.

    Journal: Scientific Reports

    Article Title: An imaging-based platform for high-content, quantitative evaluation of therapeutic response in 3D tumour models

    doi: 10.1038/srep03751

    Figure Lengend Snippet: A viability metric reports overall dose response and heterogeneity based on relative esterase activity and membrane permeability. In (a), representative thumbnails from merged images of esterase-cleaved calcein AM (green) and intercalated ethidium bromide (red) in ovarian cancer 3D overlays for (left to right), no treatment (NT) and total killing (TK) and increasing doses of paclitaxel (as a representative cytotoxic agent). Thumbnails qualitatively display increasing ethidium bromide intercalation (red) and decreasing esterase activity (green) with increasing cytotoxic dose similar for all therapies assayed here. Scale bars = 500 μm. In (b), mean fluorescence signals from full fields after rescaling ( Equation 1 ) are shown for escalating doses of paclitaxel, carboplatin, etoposide, and verteporfin PDT treatments (from left to right) in the upper plots used to quantify overall viability (lower plots, from Equation 2 ) and estimate fractional lethal doses (LD). In (c), distributions of nodule viabilities ( N > 1000 ) from automated segmentation (shown for paclitaxel-treated cultures) shifts to the left as expected with increasing dose. In (d), paclitaxel and carboplatin viability distributions at LD80 (thumbnails, left, scalebars = 500 μm) are juxtaposed to reveal a relatively tight distribution in the former in contrast to heterogeneous viability spectrum in the latter with sub-populations up to 80–90% viable.

    Article Snippet: Chemotherapy treatments Chemotherapy treatments were performed by incubating 3D cultures in growth medium with the indicated dosage of carboplatin (NDC: 61703-360-22; NOVAPLUS, Irving, TX, USA) diluted to 5–2560 μM, paclitaxel (NDC: 61703-342-22; Hospira Worldwide, Inc. Lake Forest, IL, USA), etoposide (NDC: 0703-5653-01; Teva Pharmaceuticals, Irvine, CA, USA) diluted to 1–100 μM, or gemcitabine (NDC: 0781-3282-75; Eli Lily and Company, Indianapolis, IN, USA) diluted to 0.01 to 1000 μM.

    Techniques: Activity Assay, Permeability, Fluorescence

    Size heterogeneity in vitro and in vivo and size-dependent therapeutic response. (a): In vivo nodule size heterogeneity from representative fluorescent microendoscope fields of xenograft implanatation of ovarian cancer cells, which resembles size distribution of the same cells grown in vitro. Endoscope fields are 800 μm in diameter. (b): A representation of our use of segmentation in 3D culture image data to identify individual nodules from the brightfield channel to generate a mask for computation of multiple readouts (shown in truncated form) for each individual nodule. In (c), viability-volume scatter plots output by qVISTA code showing the rescaled viability of each nodule with respect to its volume, for thousands of nodules in each treatment group, for each treatment. The vertical spread reflects dose response heterogeneity and for given a range of sizes. Probing size-dependent response in this manner reveals, for example, that the only nodules that are completely responsive to carboplatin are the smallest volume nodules while the largest remain weakly viable even at a 1000 μM (LD 80 ) dose. Response to paclitaxel is the most homogeneous and size-independent of therapies evaluated. Etoposide-treated cultures exhibit a broad spread of viabilities over a broad spread of nodule sizes, while verteporfin-PDT treatment induces a pronounced bi-modal response with simultaneously emergent populations of small nodules (1–10 cells) that are almost completely viable and almost completely non-viable.

    Journal: Scientific Reports

    Article Title: An imaging-based platform for high-content, quantitative evaluation of therapeutic response in 3D tumour models

    doi: 10.1038/srep03751

    Figure Lengend Snippet: Size heterogeneity in vitro and in vivo and size-dependent therapeutic response. (a): In vivo nodule size heterogeneity from representative fluorescent microendoscope fields of xenograft implanatation of ovarian cancer cells, which resembles size distribution of the same cells grown in vitro. Endoscope fields are 800 μm in diameter. (b): A representation of our use of segmentation in 3D culture image data to identify individual nodules from the brightfield channel to generate a mask for computation of multiple readouts (shown in truncated form) for each individual nodule. In (c), viability-volume scatter plots output by qVISTA code showing the rescaled viability of each nodule with respect to its volume, for thousands of nodules in each treatment group, for each treatment. The vertical spread reflects dose response heterogeneity and for given a range of sizes. Probing size-dependent response in this manner reveals, for example, that the only nodules that are completely responsive to carboplatin are the smallest volume nodules while the largest remain weakly viable even at a 1000 μM (LD 80 ) dose. Response to paclitaxel is the most homogeneous and size-independent of therapies evaluated. Etoposide-treated cultures exhibit a broad spread of viabilities over a broad spread of nodule sizes, while verteporfin-PDT treatment induces a pronounced bi-modal response with simultaneously emergent populations of small nodules (1–10 cells) that are almost completely viable and almost completely non-viable.

    Article Snippet: Chemotherapy treatments Chemotherapy treatments were performed by incubating 3D cultures in growth medium with the indicated dosage of carboplatin (NDC: 61703-360-22; NOVAPLUS, Irving, TX, USA) diluted to 5–2560 μM, paclitaxel (NDC: 61703-342-22; Hospira Worldwide, Inc. Lake Forest, IL, USA), etoposide (NDC: 0703-5653-01; Teva Pharmaceuticals, Irvine, CA, USA) diluted to 1–100 μM, or gemcitabine (NDC: 0781-3282-75; Eli Lily and Company, Indianapolis, IN, USA) diluted to 0.01 to 1000 μM.

    Techniques: In Vitro, In Vivo

    Contrasting quantitative reporters of cytotoxic and structural response. In (a) high-resolution 3D confocal renderings of single nodules from no treatment, carboplatin and verteporfin-PDT are shown from top to bottom to provide qualitative assessment of contrasting patterns of cytotoxic response. Scale bars = 50 μm. In (b), log-normal bi-modal nodule volume distributions show contrasting patterns between with little evidence of disruption (upper plots, given doses of etoposide and carboplatin) and treatments which with appropriate dosage appear to have a propensity to “unpack” multicellular nodules (lower plots, paclitaxel and PDT). Each histogram contains statistics on 1,000–3,000 micronodules. In the latter category there is a shift in the bimodal size distribution from the proportion of nodules in the mode corresponding to larger sizes to smaller sizes consistent with larger nodules being induced to disaggregate into an increased number of small nodules. In (c), disruption fraction, ( D frac ) based on the treatment-induced shifts in size from (B), is reported with respect to the degree of lethality for several dose for each treatment tested. Error bars show standard error. Verteporfin-PDT induces high D frac at low lethality while carboplatin and etoposide treatments suppresses viability at very high doses with negligible architectural disruption. Paclitaxel also unpacks nodules but requires higher level of cell killing in order to do so. In (d), immunofluorescence images show a clear contrast between strong E-cadherin staining at cadherens junctions in untreated nodules, with disrupted and punctate intracellular staining in PDT-treated nodules, consistent with the observation of cellular disassociation.

    Journal: Scientific Reports

    Article Title: An imaging-based platform for high-content, quantitative evaluation of therapeutic response in 3D tumour models

    doi: 10.1038/srep03751

    Figure Lengend Snippet: Contrasting quantitative reporters of cytotoxic and structural response. In (a) high-resolution 3D confocal renderings of single nodules from no treatment, carboplatin and verteporfin-PDT are shown from top to bottom to provide qualitative assessment of contrasting patterns of cytotoxic response. Scale bars = 50 μm. In (b), log-normal bi-modal nodule volume distributions show contrasting patterns between with little evidence of disruption (upper plots, given doses of etoposide and carboplatin) and treatments which with appropriate dosage appear to have a propensity to “unpack” multicellular nodules (lower plots, paclitaxel and PDT). Each histogram contains statistics on 1,000–3,000 micronodules. In the latter category there is a shift in the bimodal size distribution from the proportion of nodules in the mode corresponding to larger sizes to smaller sizes consistent with larger nodules being induced to disaggregate into an increased number of small nodules. In (c), disruption fraction, ( D frac ) based on the treatment-induced shifts in size from (B), is reported with respect to the degree of lethality for several dose for each treatment tested. Error bars show standard error. Verteporfin-PDT induces high D frac at low lethality while carboplatin and etoposide treatments suppresses viability at very high doses with negligible architectural disruption. Paclitaxel also unpacks nodules but requires higher level of cell killing in order to do so. In (d), immunofluorescence images show a clear contrast between strong E-cadherin staining at cadherens junctions in untreated nodules, with disrupted and punctate intracellular staining in PDT-treated nodules, consistent with the observation of cellular disassociation.

    Article Snippet: Chemotherapy treatments Chemotherapy treatments were performed by incubating 3D cultures in growth medium with the indicated dosage of carboplatin (NDC: 61703-360-22; NOVAPLUS, Irving, TX, USA) diluted to 5–2560 μM, paclitaxel (NDC: 61703-342-22; Hospira Worldwide, Inc. Lake Forest, IL, USA), etoposide (NDC: 0703-5653-01; Teva Pharmaceuticals, Irvine, CA, USA) diluted to 1–100 μM, or gemcitabine (NDC: 0781-3282-75; Eli Lily and Company, Indianapolis, IN, USA) diluted to 0.01 to 1000 μM.

    Techniques: Immunofluorescence, Staining

    p53 induction in response to DNA damage is impaired in TFEB/TFE3 DKO RAW264.7. 7 cells. ( A ) Representative Western blot showing p53 induction, p53 Ser15 phosphorylation, and Mdm2 levels in WT and TFE3/TFEB DKO RAW264.7 cells following etoposide treatment up to 8 hr. ( B ) Quantification of p53 induction from data shown in A. Data represents mean relative p53 level ± standard deviation with n = 3. Significance tested with two-way ANOVA with Sidak’s multiple comparisons test (**p

    Journal: eLife

    Article Title: The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage

    doi: 10.7554/eLife.40856

    Figure Lengend Snippet: p53 induction in response to DNA damage is impaired in TFEB/TFE3 DKO RAW264.7. 7 cells. ( A ) Representative Western blot showing p53 induction, p53 Ser15 phosphorylation, and Mdm2 levels in WT and TFE3/TFEB DKO RAW264.7 cells following etoposide treatment up to 8 hr. ( B ) Quantification of p53 induction from data shown in A. Data represents mean relative p53 level ± standard deviation with n = 3. Significance tested with two-way ANOVA with Sidak’s multiple comparisons test (**p

    Article Snippet: For drug treatments, cells were treated for the indicated times with the following reagents: DMSO (Sigma-Aldrich), 100 μM Etoposide (Cell Signaling Technology), dimethylformamide (Sigma-Aldrich D4551), 50 μM Cisplatin (Sigma-Aldrich 479306), Ethanol (Werner Graham Company), 2 mM LLOMe (Sigma-Aldrich L7393).

    Techniques: Western Blot, Standard Deviation

    ( A ) Representative Western blot showing TFEB and TFE3 gel shifts in response to etoposide in WT MEF, but not in p53 -/- MEF. All the immunoblots are representative of three independent experiments. ( B ) qPCR data showing relative induction of lysosomal-autophagy genes in response to starvation in WT and p53 -/- MEF. Data normalized to untreated cells and represents geometric means ± standard deviation and significance determined with Student’s t-test (*p

    Journal: eLife

    Article Title: The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage

    doi: 10.7554/eLife.40856

    Figure Lengend Snippet: ( A ) Representative Western blot showing TFEB and TFE3 gel shifts in response to etoposide in WT MEF, but not in p53 -/- MEF. All the immunoblots are representative of three independent experiments. ( B ) qPCR data showing relative induction of lysosomal-autophagy genes in response to starvation in WT and p53 -/- MEF. Data normalized to untreated cells and represents geometric means ± standard deviation and significance determined with Student’s t-test (*p

    Article Snippet: For drug treatments, cells were treated for the indicated times with the following reagents: DMSO (Sigma-Aldrich), 100 μM Etoposide (Cell Signaling Technology), dimethylformamide (Sigma-Aldrich D4551), 50 μM Cisplatin (Sigma-Aldrich 479306), Ethanol (Werner Graham Company), 2 mM LLOMe (Sigma-Aldrich L7393).

    Techniques: Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation

    TFE3 and TFEB are necessary for proper execution of apoptosis in response to DNA damage in RAW264.7 cells. ( A ) Representative Western blot showing Caspase-3 cleavage in response to increasing time of etoposide treatment. ( B ) Quantification of data shown in A indicating defects in Caspase-3 cleavage in TFEB/TFE3 DKO RAW264.7 cells. Cleaved Caspase-3 levels are normalized to WT cells after 16 hr etoposide treatment with n = 3. Significance tested with Student’s t-test (#p

    Journal: eLife

    Article Title: The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage

    doi: 10.7554/eLife.40856

    Figure Lengend Snippet: TFE3 and TFEB are necessary for proper execution of apoptosis in response to DNA damage in RAW264.7 cells. ( A ) Representative Western blot showing Caspase-3 cleavage in response to increasing time of etoposide treatment. ( B ) Quantification of data shown in A indicating defects in Caspase-3 cleavage in TFEB/TFE3 DKO RAW264.7 cells. Cleaved Caspase-3 levels are normalized to WT cells after 16 hr etoposide treatment with n = 3. Significance tested with Student’s t-test (#p

    Article Snippet: For drug treatments, cells were treated for the indicated times with the following reagents: DMSO (Sigma-Aldrich), 100 μM Etoposide (Cell Signaling Technology), dimethylformamide (Sigma-Aldrich D4551), 50 μM Cisplatin (Sigma-Aldrich 479306), Ethanol (Werner Graham Company), 2 mM LLOMe (Sigma-Aldrich L7393).

    Techniques: Western Blot

    ( A ) Immunofluorescence images of ARPE-19 cells treated with 100 μM etoposide or 50 μM Cisplatin for 24 hr or 10 hr after UVC irradiation. Scale bar = 10 μm. ( B ) Immunofluorescence images of HeLa cells treated with 100 μM etoposide for 24 hr or 50 μM Cisplatin for 12 hr and 4 hr after UVC irradiation. Scale bar = 10 μm. ( C ) Immunofluorescence images of RAW 264.7 cells treated with 100 μM etoposide for 10 hr or 35 μM Cisplatin for 10 hr or 4 hr after UVC irradiation. Scale bar = 10 μm. ( D ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in WT MEFs exposed to 100 μM etoposide for 8 hr or 50 μM Cisplatin for 10 hr or 10 hr after UVC irradiation. ( E ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in ARPE19 cells exposed to 100 μM etoposide for 24 hr or 50 μM Cisplatin for 24 hr or 24 hr after UVC irradiation. ( F ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in HeLa cells exposed to 100 μM etoposide for 24 hr or 50 μM Cisplatin for 18 hr. ( G ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in RAW 264.7 cells exposed to 100 μM etoposide for 8 hr or 50 μM Cisplatin for 8 hr or 4 hr after UVC irradiation. ( H ) Representative Western blot showing TFE3 nuclear distribution by subcellular fractionation of WT MEFs exposed to 50 μM Cisplatin for 10 hr. ( I ) Western blot showing etoposide dependent S6K de-phosphorylation in ARPE19 cells. All the immunoblots are representative of three independent experiments.

    Journal: eLife

    Article Title: The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage

    doi: 10.7554/eLife.40856

    Figure Lengend Snippet: ( A ) Immunofluorescence images of ARPE-19 cells treated with 100 μM etoposide or 50 μM Cisplatin for 24 hr or 10 hr after UVC irradiation. Scale bar = 10 μm. ( B ) Immunofluorescence images of HeLa cells treated with 100 μM etoposide for 24 hr or 50 μM Cisplatin for 12 hr and 4 hr after UVC irradiation. Scale bar = 10 μm. ( C ) Immunofluorescence images of RAW 264.7 cells treated with 100 μM etoposide for 10 hr or 35 μM Cisplatin for 10 hr or 4 hr after UVC irradiation. Scale bar = 10 μm. ( D ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in WT MEFs exposed to 100 μM etoposide for 8 hr or 50 μM Cisplatin for 10 hr or 10 hr after UVC irradiation. ( E ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in ARPE19 cells exposed to 100 μM etoposide for 24 hr or 50 μM Cisplatin for 24 hr or 24 hr after UVC irradiation. ( F ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in HeLa cells exposed to 100 μM etoposide for 24 hr or 50 μM Cisplatin for 18 hr. ( G ) Representative Western blot showing TFE3 de-phosphorylation at Ser321 and gel shift in TFEB in RAW 264.7 cells exposed to 100 μM etoposide for 8 hr or 50 μM Cisplatin for 8 hr or 4 hr after UVC irradiation. ( H ) Representative Western blot showing TFE3 nuclear distribution by subcellular fractionation of WT MEFs exposed to 50 μM Cisplatin for 10 hr. ( I ) Western blot showing etoposide dependent S6K de-phosphorylation in ARPE19 cells. All the immunoblots are representative of three independent experiments.

    Article Snippet: For drug treatments, cells were treated for the indicated times with the following reagents: DMSO (Sigma-Aldrich), 100 μM Etoposide (Cell Signaling Technology), dimethylformamide (Sigma-Aldrich D4551), 50 μM Cisplatin (Sigma-Aldrich 479306), Ethanol (Werner Graham Company), 2 mM LLOMe (Sigma-Aldrich L7393).

    Techniques: Immunofluorescence, Irradiation, Western Blot, De-Phosphorylation Assay, Electrophoretic Mobility Shift Assay, Fractionation

    ( A ) Enriched GO terms in the ‘Biological Process’ category of differentially expressed genes between etoposide-treated WT and TFEB/TFE3 DKO RAW264.7 cells. GO terms are ranked by q value (

    Journal: eLife

    Article Title: The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage

    doi: 10.7554/eLife.40856

    Figure Lengend Snippet: ( A ) Enriched GO terms in the ‘Biological Process’ category of differentially expressed genes between etoposide-treated WT and TFEB/TFE3 DKO RAW264.7 cells. GO terms are ranked by q value (

    Article Snippet: For drug treatments, cells were treated for the indicated times with the following reagents: DMSO (Sigma-Aldrich), 100 μM Etoposide (Cell Signaling Technology), dimethylformamide (Sigma-Aldrich D4551), 50 μM Cisplatin (Sigma-Aldrich 479306), Ethanol (Werner Graham Company), 2 mM LLOMe (Sigma-Aldrich L7393).

    Techniques:

    TFEB and TFE3 are essential for etoposide-induced lysosomal membrane permeabilization in MEFs. ( A ) Immunofluorescence images showing LMP in MEFs. Red galectin-1 puncta appear co-localized or within the lumen of green Lamp1 positive lysosomes. No LMP is detected under basal conditions in either WT or TFEB/TFE3 DKO MEFs. Treatment with etoposide induces profound LMP in WT, but not TFEB/TFE3 DKO cells. No differences in LMP induction were detected in LLOMe treated cells, regardless of genotype. Scale bar = 20 μm, inset = 2 μm. ( B ) WT MEFs exhibit a time-dependent increase in LMP after etoposide treatment. Quantification of data shown in A of galectin-1+/Lamp1 + LMP puncta per WT MEF cell. Data represent mean number of puncta per cell ± standard deviation from randomly selected confocal images, with > 20 cells per counted for each time point over three separate experiments. ( C ) Quantification of total number of galectin-1+/Lamp1+ LMP puncta per cell in WT vs TFE3/TFEB DKO MEFs treated for 8 hr with etoposide. Distribution is representative of one of the three independent experiments performed and shows 29 randomly selected WT MEF cells and 51 randomly selected TFEB/TFE3 DKO MEF cells. Significance determined using Student’s t-test (****p

    Journal: eLife

    Article Title: The transcription factors TFE3 and TFEB amplify p53 dependent transcriptional programs in response to DNA damage

    doi: 10.7554/eLife.40856

    Figure Lengend Snippet: TFEB and TFE3 are essential for etoposide-induced lysosomal membrane permeabilization in MEFs. ( A ) Immunofluorescence images showing LMP in MEFs. Red galectin-1 puncta appear co-localized or within the lumen of green Lamp1 positive lysosomes. No LMP is detected under basal conditions in either WT or TFEB/TFE3 DKO MEFs. Treatment with etoposide induces profound LMP in WT, but not TFEB/TFE3 DKO cells. No differences in LMP induction were detected in LLOMe treated cells, regardless of genotype. Scale bar = 20 μm, inset = 2 μm. ( B ) WT MEFs exhibit a time-dependent increase in LMP after etoposide treatment. Quantification of data shown in A of galectin-1+/Lamp1 + LMP puncta per WT MEF cell. Data represent mean number of puncta per cell ± standard deviation from randomly selected confocal images, with > 20 cells per counted for each time point over three separate experiments. ( C ) Quantification of total number of galectin-1+/Lamp1+ LMP puncta per cell in WT vs TFE3/TFEB DKO MEFs treated for 8 hr with etoposide. Distribution is representative of one of the three independent experiments performed and shows 29 randomly selected WT MEF cells and 51 randomly selected TFEB/TFE3 DKO MEF cells. Significance determined using Student’s t-test (****p

    Article Snippet: For drug treatments, cells were treated for the indicated times with the following reagents: DMSO (Sigma-Aldrich), 100 μM Etoposide (Cell Signaling Technology), dimethylformamide (Sigma-Aldrich D4551), 50 μM Cisplatin (Sigma-Aldrich 479306), Ethanol (Werner Graham Company), 2 mM LLOMe (Sigma-Aldrich L7393).

    Techniques: Immunofluorescence, Standard Deviation

    Association of CFI-400945 with conventional cytotoxic drugs doxorubicin and etoposide synergistically reduced viability of RT and MB cell lines 5nM and 10nM CFI-400945 were used in combination with doxorubicin and etoposide in concentrations ranging from 0.001 to 5μM. Viability was assessed comparing the effects of combination therapy versus treatment with each cytotoxic drug alone. Median effect plots showed that CFI-400945 in association with etoposide or doxorubicin significantly decreased the concentration of cytotoxic drug needed to affect viability of RT and MB cells (for IC50 values, please refer to Table 1 ).

    Journal: Oncotarget

    Article Title: Inhibition of polo-like kinase 4 (PLK4): a new therapeutic option for rhabdoid tumors and pediatric medulloblastoma

    doi: 10.18632/oncotarget.22704

    Figure Lengend Snippet: Association of CFI-400945 with conventional cytotoxic drugs doxorubicin and etoposide synergistically reduced viability of RT and MB cell lines 5nM and 10nM CFI-400945 were used in combination with doxorubicin and etoposide in concentrations ranging from 0.001 to 5μM. Viability was assessed comparing the effects of combination therapy versus treatment with each cytotoxic drug alone. Median effect plots showed that CFI-400945 in association with etoposide or doxorubicin significantly decreased the concentration of cytotoxic drug needed to affect viability of RT and MB cells (for IC50 values, please refer to Table 1 ).

    Article Snippet: Compounds Cells were treated as indicated below with the following compounds: CFI-400945 (CAS 1338800-06-8 – Cayman Chemical, USA), doxorubicin (CAS 25316-40-9 – MedChem Express, USA) and etoposide (CAS 33419-42-0 – Cayman Chemical, USA).

    Techniques: Concentration Assay

    The lncRNA transcription rates were not affected by chemical stressors. The transcription rates of the lncRNAs ( a ) OIP5-AS1, ( b ) FLJ46906, ( c ) LINC00137, and ( d ) GABPB1-AS1 were examined in control cells (black bar) and those exposed to hydrogen peroxide (gray bar), mercury II chloride (pale gray bar), or etoposide (white bar) as chemical stressors. The nascent lncRNAs incorporated EU during transcription, and the relative EU-RNA quantity reflects the total amount of EU-labeled RNA captured divided by the input amount of RNA as an indicator of the transcription rate. All values are means ± SD from three independent experiments.

    Journal: Scientific Reports

    Article Title: Short-lived long noncoding RNAs as surrogate indicators for chemical stress in HepG2 cells and their degradation by nuclear RNases

    doi: 10.1038/s41598-019-56869-y

    Figure Lengend Snippet: The lncRNA transcription rates were not affected by chemical stressors. The transcription rates of the lncRNAs ( a ) OIP5-AS1, ( b ) FLJ46906, ( c ) LINC00137, and ( d ) GABPB1-AS1 were examined in control cells (black bar) and those exposed to hydrogen peroxide (gray bar), mercury II chloride (pale gray bar), or etoposide (white bar) as chemical stressors. The nascent lncRNAs incorporated EU during transcription, and the relative EU-RNA quantity reflects the total amount of EU-labeled RNA captured divided by the input amount of RNA as an indicator of the transcription rate. All values are means ± SD from three independent experiments.

    Article Snippet: Etoposide was purchased from BioVision (Mountain View, CA).

    Techniques: Labeling

    The decay rates of lncRNAs were reduced by chemical stressors. The rates of decay of the lncRNAs ( a ) OIP5-AS1, ( b ) FLJ46906, ( c) LINC00137, and ( d ) GABPB1-AS1 were examined in control cells (solid circles and black lines) and those exposed to hydrogen peroxide (open circles/gray line), mercury II chloride (solid squares/black dotted line), or etoposide (open squares/gray dotted line) as chemical stressors. All values are means ± SD from three independent experiments.

    Journal: Scientific Reports

    Article Title: Short-lived long noncoding RNAs as surrogate indicators for chemical stress in HepG2 cells and their degradation by nuclear RNases

    doi: 10.1038/s41598-019-56869-y

    Figure Lengend Snippet: The decay rates of lncRNAs were reduced by chemical stressors. The rates of decay of the lncRNAs ( a ) OIP5-AS1, ( b ) FLJ46906, ( c) LINC00137, and ( d ) GABPB1-AS1 were examined in control cells (solid circles and black lines) and those exposed to hydrogen peroxide (open circles/gray line), mercury II chloride (solid squares/black dotted line), or etoposide (open squares/gray dotted line) as chemical stressors. All values are means ± SD from three independent experiments.

    Article Snippet: Etoposide was purchased from BioVision (Mountain View, CA).

    Techniques:

    Exposure to chemical stressors altered the levels of lncRNA expression. Following treatment of HepG2 cells with 100 μM ( a ) hydrogen peroxide, ( b ) mercury II chloride, or ( c ) etoposide for 24 hours, RT-qPCR was performed to determine the expression levels of the indicated RNAs normalized relative to GAPDH, ACTB, HPRT1, and PGK1. All values are means ± SD from four independent experiments (* P

    Journal: Scientific Reports

    Article Title: Short-lived long noncoding RNAs as surrogate indicators for chemical stress in HepG2 cells and their degradation by nuclear RNases

    doi: 10.1038/s41598-019-56869-y

    Figure Lengend Snippet: Exposure to chemical stressors altered the levels of lncRNA expression. Following treatment of HepG2 cells with 100 μM ( a ) hydrogen peroxide, ( b ) mercury II chloride, or ( c ) etoposide for 24 hours, RT-qPCR was performed to determine the expression levels of the indicated RNAs normalized relative to GAPDH, ACTB, HPRT1, and PGK1. All values are means ± SD from four independent experiments (* P

    Article Snippet: Etoposide was purchased from BioVision (Mountain View, CA).

    Techniques: Expressing, Quantitative RT-PCR

    Chemical stressors changed lncRNA expression levels. Following treatment of HepG2 cells with 100 μM ( a ) hydrogen peroxide, ( b ) mercury II chloride, or ( c ) etoposide for 24 hours, RT-qPCR was performed to determine the expression levels of the indicated RNAs normalized relative to GAPDH, ACTB, HPRT1, and PGK1. All values are means ± SD from four independent experiments (* P

    Journal: Scientific Reports

    Article Title: Short-lived long noncoding RNAs as surrogate indicators for chemical stress in HepG2 cells and their degradation by nuclear RNases

    doi: 10.1038/s41598-019-56869-y

    Figure Lengend Snippet: Chemical stressors changed lncRNA expression levels. Following treatment of HepG2 cells with 100 μM ( a ) hydrogen peroxide, ( b ) mercury II chloride, or ( c ) etoposide for 24 hours, RT-qPCR was performed to determine the expression levels of the indicated RNAs normalized relative to GAPDH, ACTB, HPRT1, and PGK1. All values are means ± SD from four independent experiments (* P

    Article Snippet: Etoposide was purchased from BioVision (Mountain View, CA).

    Techniques: Expressing, Quantitative RT-PCR

    Perifosine increased the sensitivity of high TrkB expressing TB3 tumors to etoposide treatment. A. Mice were treated with vehicle or perifosine at 10mg/kg, 15mg/kg or 20mg/kg for 30 days. The tumor size in treated groups was compared to that of control group. # P

    Journal: Cancer

    Article Title: Perifosine-induced inhibition of Akt attenuates BDNF/TrkB-induced chemoresistance in neuroblastoma in vivo

    doi: 10.1002/cncr.26133

    Figure Lengend Snippet: Perifosine increased the sensitivity of high TrkB expressing TB3 tumors to etoposide treatment. A. Mice were treated with vehicle or perifosine at 10mg/kg, 15mg/kg or 20mg/kg for 30 days. The tumor size in treated groups was compared to that of control group. # P

    Article Snippet: Etoposide was obtained from Bedford Laboratories (Bedford, OH).

    Techniques: Expressing, Mouse Assay

    High TrkB expressing tumors are less sensitive to etoposide than low TrkB expressing tumors. Mice with tumors were treated with vehicle or etoposide at 10mg/kg or 20mg/kg for 21days. The size of the tumors from the etoposide-treated groups were compared to that of control group. # P

    Journal: Cancer

    Article Title: Perifosine-induced inhibition of Akt attenuates BDNF/TrkB-induced chemoresistance in neuroblastoma in vivo

    doi: 10.1002/cncr.26133

    Figure Lengend Snippet: High TrkB expressing tumors are less sensitive to etoposide than low TrkB expressing tumors. Mice with tumors were treated with vehicle or etoposide at 10mg/kg or 20mg/kg for 21days. The size of the tumors from the etoposide-treated groups were compared to that of control group. # P

    Article Snippet: Etoposide was obtained from Bedford Laboratories (Bedford, OH).

    Techniques: Expressing, Mouse Assay

    Perifosine increased the sensitivity of NGP tumors to etoposide treatment. A: Proteins from NGP, LAN5 and SY5Y cells were extracted and expressions of TrkB, GAPDH, P-Akt (Ser473) and T-Akt were analyzed by Western Blotting. B, C, D: NGP tumors were treated with etoposide (10mg/kg, 20mg/kg) (B) or perifosine (10mg/kg, 17mg/kg, 24mg/kg)(C) individually, or a combination of etopsoide (10mg/kg) and perifosine(17mg/kg) (D). The tumor size in treated groups was compared to that of control group. B: * P

    Journal: Cancer

    Article Title: Perifosine-induced inhibition of Akt attenuates BDNF/TrkB-induced chemoresistance in neuroblastoma in vivo

    doi: 10.1002/cncr.26133

    Figure Lengend Snippet: Perifosine increased the sensitivity of NGP tumors to etoposide treatment. A: Proteins from NGP, LAN5 and SY5Y cells were extracted and expressions of TrkB, GAPDH, P-Akt (Ser473) and T-Akt were analyzed by Western Blotting. B, C, D: NGP tumors were treated with etoposide (10mg/kg, 20mg/kg) (B) or perifosine (10mg/kg, 17mg/kg, 24mg/kg)(C) individually, or a combination of etopsoide (10mg/kg) and perifosine(17mg/kg) (D). The tumor size in treated groups was compared to that of control group. B: * P

    Article Snippet: Etoposide was obtained from Bedford Laboratories (Bedford, OH).

    Techniques: Western Blot

    TrkB expression is regulated by tetracycline in vitro and affects the NB cell response to chemotherapy. TB3 cells were cultured for 3 days in the presence or absence of tetracycline (1μg/ml), then total RNA was extracted (A); treated with BDNF (100ng/ml) for 15min, then total protein was extracted (B, C), treated with etoposide (D) or a combination of BDNF and etoposide (E, F) for 24hrs. A. Total RNA (1μg) was reverse-transcribed into cDNA following by quantitative-PCR analysis for TrkB mRNA. B. Protein lysates (500μg/ml) were immunoprecipitated with anti-pan Trk antibody and subjected to Western Blotting for evaluation of TrkB and phosphorylated tyrosine. C. Total protein (30μg) were immunoblotted for P-Akt (Ser473), P-Akt (Thr308) and total Akt. In vitro Akt kinase activity was assessed by immunoprecipitating with P-Akt (Ser473) antibody, and then using GSK-3 as a substrate to measure Akt activity indicated by phosphorylated GSK-3α/β (Ser21/9). D, E and F : MTS assay was used to determine cell survival at the end of treatment. * P

    Journal: Cancer

    Article Title: Perifosine-induced inhibition of Akt attenuates BDNF/TrkB-induced chemoresistance in neuroblastoma in vivo

    doi: 10.1002/cncr.26133

    Figure Lengend Snippet: TrkB expression is regulated by tetracycline in vitro and affects the NB cell response to chemotherapy. TB3 cells were cultured for 3 days in the presence or absence of tetracycline (1μg/ml), then total RNA was extracted (A); treated with BDNF (100ng/ml) for 15min, then total protein was extracted (B, C), treated with etoposide (D) or a combination of BDNF and etoposide (E, F) for 24hrs. A. Total RNA (1μg) was reverse-transcribed into cDNA following by quantitative-PCR analysis for TrkB mRNA. B. Protein lysates (500μg/ml) were immunoprecipitated with anti-pan Trk antibody and subjected to Western Blotting for evaluation of TrkB and phosphorylated tyrosine. C. Total protein (30μg) were immunoblotted for P-Akt (Ser473), P-Akt (Thr308) and total Akt. In vitro Akt kinase activity was assessed by immunoprecipitating with P-Akt (Ser473) antibody, and then using GSK-3 as a substrate to measure Akt activity indicated by phosphorylated GSK-3α/β (Ser21/9). D, E and F : MTS assay was used to determine cell survival at the end of treatment. * P

    Article Snippet: Etoposide was obtained from Bedford Laboratories (Bedford, OH).

    Techniques: Expressing, In Vitro, Cell Culture, Real-time Polymerase Chain Reaction, Immunoprecipitation, Western Blot, Activity Assay, MTS Assay

    p53 R175H and MDM2 proteins synergistically reduce chemosensitivity of lung and breast cancer cells ( A ) H1299-R175H-MDM2 cell line was treated with Ponasterone A (Pon A) and/or Doxycycline (Dox) for 24 h to induce p53 R175H and/or MDM2, respectively. Immunoblotting with specific antibody revealed tight and efficient expression of both proteins. ( B ) Induced and uninduced cells were grown in triplicate in chambers compatible with the xCELLigence RTCA DP Instrument and Cisplatin (40 μM) was added at the indicated time point. Proliferative index was monitored for 120 h. Mean and standard deviation of three repeats are shown. ( C ) After 48 h treatment with 60 μM Cisplatin (left panel) or Etoposide (right panel), the apoptotic response of induced or uninduced cells stained with Annexin V/Gel Green dye was measured with a flow cytometer. p53 R175H or MDM2 expressed alone reduced apoptosis to same extent, whereas significant decrease was observed after simultaneous induction of both proteins. Bars represent the relative decrease (%) of cells in early apoptosis (Annexin V positive, Gel Green dye negative), estimated as follows = ( Value − Baseline ) Baseline × 100 (Baseline–Apoptotic response of uninduced H1299-R175H-MDM2 cell line). Statistical significance ( P value) was counted for three independent experiments with Anova statistical test. *, **, ***, **** indicate statistical significance p

    Journal: Oncotarget

    Article Title: Molecular chaperones in the acquisition of cancer cell chemoresistance with mutated TP53 and MDM2 up-regulation

    doi: 10.18632/oncotarget.18899

    Figure Lengend Snippet: p53 R175H and MDM2 proteins synergistically reduce chemosensitivity of lung and breast cancer cells ( A ) H1299-R175H-MDM2 cell line was treated with Ponasterone A (Pon A) and/or Doxycycline (Dox) for 24 h to induce p53 R175H and/or MDM2, respectively. Immunoblotting with specific antibody revealed tight and efficient expression of both proteins. ( B ) Induced and uninduced cells were grown in triplicate in chambers compatible with the xCELLigence RTCA DP Instrument and Cisplatin (40 μM) was added at the indicated time point. Proliferative index was monitored for 120 h. Mean and standard deviation of three repeats are shown. ( C ) After 48 h treatment with 60 μM Cisplatin (left panel) or Etoposide (right panel), the apoptotic response of induced or uninduced cells stained with Annexin V/Gel Green dye was measured with a flow cytometer. p53 R175H or MDM2 expressed alone reduced apoptosis to same extent, whereas significant decrease was observed after simultaneous induction of both proteins. Bars represent the relative decrease (%) of cells in early apoptosis (Annexin V positive, Gel Green dye negative), estimated as follows = ( Value − Baseline ) Baseline × 100 (Baseline–Apoptotic response of uninduced H1299-R175H-MDM2 cell line). Statistical significance ( P value) was counted for three independent experiments with Anova statistical test. *, **, ***, **** indicate statistical significance p

    Article Snippet: Cells were treated with Ponasterone A (Invitrogen), Doxycycline (Sigma-Aldrich), Cisplatin (Tocris Bioscience), Camptothecin (Selleck Chemicals), Doxorubicin (Tocris Bioscience), Etoposide (Tocris Bioscience), Taxol (Tocris Bioscience), at the following concentrations: 0.5–3 µM, 50 ng/ml, 10–80 µM, 1–5 µM, 0.25–2.5 µM, 40–80 μM, 0.1–0.5 μM, respectively.

    Techniques: Expressing, Standard Deviation, Staining, Flow Cytometry, Cytometry

    DNA damage induces de-phosphorylation of nuclear Tau. ( a ) The effect of Etoposide and Vinblastine treatment on mouse C17.2 cells is shown by confocal microscopy upon immune staining of PFA-fixed cells with antibodies against the microtubule marker β-tubulin (in cyan) or the DNA damage marker γH2A-X (in red). ( b ) Confocal microscopic quantification of the activated kinases in the nucleus (DAPI mask). Mean percent ± sem relative to the respective controls. 2-tailed unpaired Mann-Whitney test, ****p

    Journal: Scientific Reports

    Article Title: Phosphorylation of nuclear Tau is modulated by distinct cellular pathways

    doi: 10.1038/s41598-018-36374-4

    Figure Lengend Snippet: DNA damage induces de-phosphorylation of nuclear Tau. ( a ) The effect of Etoposide and Vinblastine treatment on mouse C17.2 cells is shown by confocal microscopy upon immune staining of PFA-fixed cells with antibodies against the microtubule marker β-tubulin (in cyan) or the DNA damage marker γH2A-X (in red). ( b ) Confocal microscopic quantification of the activated kinases in the nucleus (DAPI mask). Mean percent ± sem relative to the respective controls. 2-tailed unpaired Mann-Whitney test, ****p

    Article Snippet: Drug treatments During the last 5 h of tetracycline incubation, C17.2 cells with inducible Tau expression were treated with 60 µM Etoposide (Abcam, ab120227; 100 mM stock in DMSO), 3 µM Vinblastine (Sigma-Aldrich, V1377; 11 mM stock in DMSO), or 60 nM Leptomycin B (Sigma-Aldrich, L2913; 10.3 µM in 70% ethanol).

    Techniques: De-Phosphorylation Assay, Confocal Microscopy, Staining, Marker, MANN-WHITNEY

    Characteristics of H1299 and A549  VR  cells. (A) H1299 parental and  VR  (weak, moderate, and strong resistant) cell viabilities in response to  VRB . H1299 parental and  VR  cells were treated for 96 h with increasing concentrations of  VRB . Cell viability was determined using the  WST ‐8 assay and is shown as a percentage of the value of the untreated cells. The sigmoid curves were drawn using Prism software. Experiments were performed in triplicate. (B) The  IC 50 values for  VRB ,  PAC ,  DOC ,  VP ‐16, and  CDDP  in H1299 parental and  VR  cells. Cell viability in response to  PAC ,  DOC ,  VP ‐16, and  CDDP  was measured as described for  VRB . Experiments were performed in triplicate. The  IC 50 values were calculated using Prism software. The error bars show the 95%  CI . *  P

    Journal: Cancer Medicine

    Article Title: The synergistic role of ATP‐dependent drug efflux pump and focal adhesion signaling pathways in vinorelbine resistance in lung cancer

    doi: 10.1002/cam4.1282

    Figure Lengend Snippet: Characteristics of H1299 and A549 VR cells. (A) H1299 parental and VR (weak, moderate, and strong resistant) cell viabilities in response to VRB . H1299 parental and VR cells were treated for 96 h with increasing concentrations of VRB . Cell viability was determined using the WST ‐8 assay and is shown as a percentage of the value of the untreated cells. The sigmoid curves were drawn using Prism software. Experiments were performed in triplicate. (B) The IC 50 values for VRB , PAC , DOC , VP ‐16, and CDDP in H1299 parental and VR cells. Cell viability in response to PAC , DOC , VP ‐16, and CDDP was measured as described for VRB . Experiments were performed in triplicate. The IC 50 values were calculated using Prism software. The error bars show the 95% CI . * P

    Article Snippet: Compounds VRB, cisplatin, paclitaxel, docetaxel, and etoposide were purchased from Wako Pure Chemical Industries (Osaka, Japan).

    Techniques: Software

    Etoposide-induced DSB repair by NHEJ involves the MRN complex. γH2AX foci kinetics were assessed in primary human fibroblasts. Mre11-defective (ATLD2) and Nbs1-defective (CZD82CH and GM07166A) but not ATM-defective primary human fibroblasts (AT1BR) exhibit elevated foci levels after 20 µM etoposide treatment in G0/G1 phase. Background foci numbers were subtracted. Error bars represent the SD from at least three different experiments.

    Journal: Nucleic Acids Research

    Article Title: CtIP and MRN promote non-homologous end-joining of etoposide-induced DNA double-strand breaks in G1

    doi: 10.1093/nar/gkq1175

    Figure Lengend Snippet: Etoposide-induced DSB repair by NHEJ involves the MRN complex. γH2AX foci kinetics were assessed in primary human fibroblasts. Mre11-defective (ATLD2) and Nbs1-defective (CZD82CH and GM07166A) but not ATM-defective primary human fibroblasts (AT1BR) exhibit elevated foci levels after 20 µM etoposide treatment in G0/G1 phase. Background foci numbers were subtracted. Error bars represent the SD from at least three different experiments.

    Article Snippet: Aclarubicin at 5 µM was added immediately before etoposide treatment.

    Techniques: Non-Homologous End Joining

    Etoposide-induced DSBs are repaired by NHEJ. ( A ) HSF1 cells were incubated with a specific ATM and DNA-PK inhibitor 1 h prior to etoposide treatment or irradiation. Foci formation is abolished by combined inhibitor treatment, showing that the kinases ATM and DNA-PK but not ATR phosphorylate H2AX. ( B ) γH2AX foci due to etoposide (etopo) treatment require topoII activity. Pre-treatment with aclarubicin (acl.), a topoII inhibitor, abolishes the formation of etoposide-induced γH2AX foci. Aclarubicin alone does not form γH2AX foci. ( C ) γH2AX foci kinetics in primary human fibroblasts. Wt (HSF1 and C2886) and Brca2-deficient cells (HSC62) show similar repair kinetics whereas LigIV-deficient cells (180 BR) exhibit elevated γH2AX foci levels after 20 µM etoposide treatment in G0/G1. Background foci numbers were subtracted. Error bars represent the standard deviation (SD) from at least three different experiments.

    Journal: Nucleic Acids Research

    Article Title: CtIP and MRN promote non-homologous end-joining of etoposide-induced DNA double-strand breaks in G1

    doi: 10.1093/nar/gkq1175

    Figure Lengend Snippet: Etoposide-induced DSBs are repaired by NHEJ. ( A ) HSF1 cells were incubated with a specific ATM and DNA-PK inhibitor 1 h prior to etoposide treatment or irradiation. Foci formation is abolished by combined inhibitor treatment, showing that the kinases ATM and DNA-PK but not ATR phosphorylate H2AX. ( B ) γH2AX foci due to etoposide (etopo) treatment require topoII activity. Pre-treatment with aclarubicin (acl.), a topoII inhibitor, abolishes the formation of etoposide-induced γH2AX foci. Aclarubicin alone does not form γH2AX foci. ( C ) γH2AX foci kinetics in primary human fibroblasts. Wt (HSF1 and C2886) and Brca2-deficient cells (HSC62) show similar repair kinetics whereas LigIV-deficient cells (180 BR) exhibit elevated γH2AX foci levels after 20 µM etoposide treatment in G0/G1. Background foci numbers were subtracted. Error bars represent the standard deviation (SD) from at least three different experiments.

    Article Snippet: Aclarubicin at 5 µM was added immediately before etoposide treatment.

    Techniques: Non-Homologous End Joining, Incubation, Irradiation, Activity Assay, Standard Deviation

    NHEJ of etoposide-induced DSBs in G1/G0 involves CtIP. ( A ) Identification of cell cycle phases in HeLa cells (see text for explanation). ( B ) γH2AX foci kinetics in siRNA treated HeLa cells analyzed 48 h after transfection. Down-regulation of Mre11 alone, CtIP alone or Mre11 and CtIP in combination results in similarly elevated γH2AX foci levels after etoposide treatment in G1-phase cells. Background foci numbers were subtracted. Error bars represent the SD from at least three different experiments. ( C ) γH2AX foci kinetics in hTert immortalized human fibroblasts. CtIP down-regulation in wt cells (82-6 hTert) results in a modest but significant repair defect. XLF-deficient cells (2BN hTert) exhibit a substantially higher repair defect. CtIP depletion in XLF-defective cells has no additional effect. Efficient CtIP down-regulation was confirmed by the abolishment of Rad51 foci formation after irradiation (data not shown). Background foci numbers were subtracted. Error bars represent the SD from at least three different experiments.

    Journal: Nucleic Acids Research

    Article Title: CtIP and MRN promote non-homologous end-joining of etoposide-induced DNA double-strand breaks in G1

    doi: 10.1093/nar/gkq1175

    Figure Lengend Snippet: NHEJ of etoposide-induced DSBs in G1/G0 involves CtIP. ( A ) Identification of cell cycle phases in HeLa cells (see text for explanation). ( B ) γH2AX foci kinetics in siRNA treated HeLa cells analyzed 48 h after transfection. Down-regulation of Mre11 alone, CtIP alone or Mre11 and CtIP in combination results in similarly elevated γH2AX foci levels after etoposide treatment in G1-phase cells. Background foci numbers were subtracted. Error bars represent the SD from at least three different experiments. ( C ) γH2AX foci kinetics in hTert immortalized human fibroblasts. CtIP down-regulation in wt cells (82-6 hTert) results in a modest but significant repair defect. XLF-deficient cells (2BN hTert) exhibit a substantially higher repair defect. CtIP depletion in XLF-defective cells has no additional effect. Efficient CtIP down-regulation was confirmed by the abolishment of Rad51 foci formation after irradiation (data not shown). Background foci numbers were subtracted. Error bars represent the SD from at least three different experiments.

    Article Snippet: Aclarubicin at 5 µM was added immediately before etoposide treatment.

    Techniques: Non-Homologous End Joining, Transfection, Irradiation

    CtIP function during repair of etoposide-induced DSBs in G1 requires Thr-847 phosphorylation. ( A ) HeLa cells were depleted for endogenous CtIP by siRNA and transfected with various GFP-tagged CtIP plasmids. Only GFP-positive cells in G1 were analyzed. ( B ) γH2AX foci kinetics in HeLa cells after 20 µM etoposide. Cells transfected with the CtIP mutation T847A but not the mutation S327A exhibit a repair defect. Background foci numbers were subtracted. Error bars represent the SD from at least three different experiments. ( C ) γH2AX foci kinetics in HeLa cells after 100 µM etoposide. Background foci numbers were subtracted. Error bars represent the SD from at least two different experiments. ( D ) Rad51 foci in CENP-F positive G2-phase HeLa cells after 2 Gy X-rays. Cells transfected with the CtIP mutation T847A or the mutation S327A exhibit a defect in the formation of Rad51 foci. Background foci numbers were subtracted. Error bars represent the SD from at least two different experiments. ( E ) γH2AX and Rad51 foci analysis in HeLa cells treated with the CDK inhibitor roscovitine (rosc) for 3 h prior to etoposide treatment or irradiation. CDK inhibition does not affect γH2AX foci levels after etoposide treatment in G1-phase cells but inhibits Rad51 foci formation after 2 Gy X-irradiation in G2-phase cells. Background foci numbers were subtracted. Error bars represent the SD from at least two different experiments.

    Journal: Nucleic Acids Research

    Article Title: CtIP and MRN promote non-homologous end-joining of etoposide-induced DNA double-strand breaks in G1

    doi: 10.1093/nar/gkq1175

    Figure Lengend Snippet: CtIP function during repair of etoposide-induced DSBs in G1 requires Thr-847 phosphorylation. ( A ) HeLa cells were depleted for endogenous CtIP by siRNA and transfected with various GFP-tagged CtIP plasmids. Only GFP-positive cells in G1 were analyzed. ( B ) γH2AX foci kinetics in HeLa cells after 20 µM etoposide. Cells transfected with the CtIP mutation T847A but not the mutation S327A exhibit a repair defect. Background foci numbers were subtracted. Error bars represent the SD from at least three different experiments. ( C ) γH2AX foci kinetics in HeLa cells after 100 µM etoposide. Background foci numbers were subtracted. Error bars represent the SD from at least two different experiments. ( D ) Rad51 foci in CENP-F positive G2-phase HeLa cells after 2 Gy X-rays. Cells transfected with the CtIP mutation T847A or the mutation S327A exhibit a defect in the formation of Rad51 foci. Background foci numbers were subtracted. Error bars represent the SD from at least two different experiments. ( E ) γH2AX and Rad51 foci analysis in HeLa cells treated with the CDK inhibitor roscovitine (rosc) for 3 h prior to etoposide treatment or irradiation. CDK inhibition does not affect γH2AX foci levels after etoposide treatment in G1-phase cells but inhibits Rad51 foci formation after 2 Gy X-irradiation in G2-phase cells. Background foci numbers were subtracted. Error bars represent the SD from at least two different experiments.

    Article Snippet: Aclarubicin at 5 µM was added immediately before etoposide treatment.

    Techniques: Transfection, Mutagenesis, Irradiation, Inhibition

    Sensitivity of primary and transformed cells to DNA damaging agents in the presence and absence of C9orf82 protein. (A) Relative survival of pre-B cells from wild type and C9orf82 ko/ko mice upon exposure to increasing doses of UVC, cisplatin, MMS, γ-irradiation, doxorubicin and etoposide was analyzed. (B) Colony survival rate of transformed MEFs from wild type and C9orf82 ko/ko mice upon exposure to Doxorubicin and Etoposide was compared. (C) Recovery from DNA damage response. The recovery rate from γH2AX generated in Tp53kd transformed MEFs in response to etoposide induced DSBs are compared. (D) Cell cycle analysis and relative contribution of sub-G, G1, and S/G2 of pre-B cells from the indicted genotypes.

    Journal: PLoS ONE

    Article Title: Towards an understanding of C9orf82 protein/CAAP1 function

    doi: 10.1371/journal.pone.0210526

    Figure Lengend Snippet: Sensitivity of primary and transformed cells to DNA damaging agents in the presence and absence of C9orf82 protein. (A) Relative survival of pre-B cells from wild type and C9orf82 ko/ko mice upon exposure to increasing doses of UVC, cisplatin, MMS, γ-irradiation, doxorubicin and etoposide was analyzed. (B) Colony survival rate of transformed MEFs from wild type and C9orf82 ko/ko mice upon exposure to Doxorubicin and Etoposide was compared. (C) Recovery from DNA damage response. The recovery rate from γH2AX generated in Tp53kd transformed MEFs in response to etoposide induced DSBs are compared. (D) Cell cycle analysis and relative contribution of sub-G, G1, and S/G2 of pre-B cells from the indicted genotypes.

    Article Snippet: Colony survival MEFs MEFs were seeded in 10 cm dishes at a density of 200 cells/dish in complete medium with varying concentrations of Doxorubicin or Etoposide (Pharmachemie).

    Techniques: Transformation Assay, Mouse Assay, Irradiation, Generated, Cell Cycle Assay

    Active caspase-3 level in C9orf82 protein proficient and deficient cells in response to Doxorubicin and Etoposide. (A) The percentage of active Caspase-3 without prior exposure to Doxorubicin and Etoposide was compared between the polyclonal pre-B cell cultures established from the fetal livers of two independent E14.5 embryos per genotype. (B) Relative contribution of Doxorubicin and Etoposide exposure to the level of active Caspase-3 was analyzed between the fetal liver derived pre-B cell cultures from two independent E14.5 embryos per genotype.

    Journal: PLoS ONE

    Article Title: Towards an understanding of C9orf82 protein/CAAP1 function

    doi: 10.1371/journal.pone.0210526

    Figure Lengend Snippet: Active caspase-3 level in C9orf82 protein proficient and deficient cells in response to Doxorubicin and Etoposide. (A) The percentage of active Caspase-3 without prior exposure to Doxorubicin and Etoposide was compared between the polyclonal pre-B cell cultures established from the fetal livers of two independent E14.5 embryos per genotype. (B) Relative contribution of Doxorubicin and Etoposide exposure to the level of active Caspase-3 was analyzed between the fetal liver derived pre-B cell cultures from two independent E14.5 embryos per genotype.

    Article Snippet: Colony survival MEFs MEFs were seeded in 10 cm dishes at a density of 200 cells/dish in complete medium with varying concentrations of Doxorubicin or Etoposide (Pharmachemie).

    Techniques: Derivative Assay

    Tipifarnib enhances induction of apoptosis by etoposide in AML cell lines . (A) Log-phase HL-60 cells were treated for 24 hours with diluent, 1.5 μM etoposide, 1 μM tipifarnib, or the combination of 1.5 μM etoposide plus 1 μM

    Journal: Blood

    Article Title: Active oral regimen for elderly adults with newly diagnosed acute myelogenous leukemia: a preclinical and phase 1 trial of the farnesyltransferase inhibitor tipifarnib (R115777, Zarnestra) combined with etoposide

    doi: 10.1182/blood-2008-08-172726

    Figure Lengend Snippet: Tipifarnib enhances induction of apoptosis by etoposide in AML cell lines . (A) Log-phase HL-60 cells were treated for 24 hours with diluent, 1.5 μM etoposide, 1 μM tipifarnib, or the combination of 1.5 μM etoposide plus 1 μM

    Article Snippet: Tipifarnib was provided by David End (Johnson & Johnson, Springhouse, PA); and etoposide was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA).

    Techniques:

    Effect of tipifarnib on antiproliferative effects of etoposide in clinical AML isolates . (A-C) Freshly isolated mononuclear cells from 3 newly diagnosed AML patients were plated in methylcellulose in diluent, 25 nM tipifarnib plus 50 to 200 nM etoposide,

    Journal: Blood

    Article Title: Active oral regimen for elderly adults with newly diagnosed acute myelogenous leukemia: a preclinical and phase 1 trial of the farnesyltransferase inhibitor tipifarnib (R115777, Zarnestra) combined with etoposide

    doi: 10.1182/blood-2008-08-172726

    Figure Lengend Snippet: Effect of tipifarnib on antiproliferative effects of etoposide in clinical AML isolates . (A-C) Freshly isolated mononuclear cells from 3 newly diagnosed AML patients were plated in methylcellulose in diluent, 25 nM tipifarnib plus 50 to 200 nM etoposide,

    Article Snippet: Tipifarnib was provided by David End (Johnson & Johnson, Springhouse, PA); and etoposide was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA).

    Techniques: Isolation

    Estimated dose-response relationship between combinations of etoposide and tipifarnib . The height of the surface and its shading indicate the response rate for each dose combination.

    Journal: Blood

    Article Title: Active oral regimen for elderly adults with newly diagnosed acute myelogenous leukemia: a preclinical and phase 1 trial of the farnesyltransferase inhibitor tipifarnib (R115777, Zarnestra) combined with etoposide

    doi: 10.1182/blood-2008-08-172726

    Figure Lengend Snippet: Estimated dose-response relationship between combinations of etoposide and tipifarnib . The height of the surface and its shading indicate the response rate for each dose combination.

    Article Snippet: Tipifarnib was provided by David End (Johnson & Johnson, Springhouse, PA); and etoposide was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA).

    Techniques:

    Antiproliferative effects of tipifarnib-containing combinations . (A,B) Log-phase HL-60 cells were treated with the indicated concentration of etoposide (○, A), tipifarnib (○, B), or the combination at a 2:1 ratio (●, A,B). (C)

    Journal: Blood

    Article Title: Active oral regimen for elderly adults with newly diagnosed acute myelogenous leukemia: a preclinical and phase 1 trial of the farnesyltransferase inhibitor tipifarnib (R115777, Zarnestra) combined with etoposide

    doi: 10.1182/blood-2008-08-172726

    Figure Lengend Snippet: Antiproliferative effects of tipifarnib-containing combinations . (A,B) Log-phase HL-60 cells were treated with the indicated concentration of etoposide (○, A), tipifarnib (○, B), or the combination at a 2:1 ratio (●, A,B). (C)

    Article Snippet: Tipifarnib was provided by David End (Johnson & Johnson, Springhouse, PA); and etoposide was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA).

    Techniques: Concentration Assay