etoposide Millipore Search Results


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  • 94
    Millipore etoposide
    Validation of iTRAQ data through Western blot analysis. H2AX was selected for validation of the expression alteration trends through Western blot analysis. The results showed that H2AX is downregulated in Caki-1 cells compared with XIAP knockdown cells after <t>etoposide</t> treatment. GAPDH was used as a loading control. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; XIAP: X-linked inhibitor of apoptosis.
    Etoposide, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 8007 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore doxorubicin
    Interleukin 11 (IL-11)/IL-11R signaling pathway induced the chemo-resistance through JAK/STAT3 pathway. (A) Immunofluoresence of p-JAK2 in BGC823 and SGC7901 cells pre-treated with or without cultured medium of cancer-associated-fibroblasts (CAFs-CM)/rhIL-11 in the presence or absence of IL-11 neutralizing antibody. (B) Immunofluoresence of p-STAT3 in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (C) Western blotting of p-JAK2, total JAK2 and β-actin in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (D) Western blotting of p-STAT3, total STAT3, and β-actin in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (E) The cell viability of BGC823 cells treated with 6 μg/mL DDP, 6 μM etoposide, and 6 μM <t>doxorubicin</t> respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of ruxolitinib (5 μM). PBS, phosphate buffered saline; Dox, doxorubicin; Eto, etoposide. (F) The cell viability of SGC7901 cells treated with 4 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of ruxolitinib (5 μM). (G) The cell viability of BGC823 cells treated with 6 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the wild type or shSTAT3 cells. (H) The cell viability of SGC7901 cells treated with 4 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the wild type or shSTAT3 cells. The data was presented as the mean±standard error of mean from three independent experiments. ** p
    Doxorubicin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 8784 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore cisplatin
    Synergistic effects of A-443654 on <t>cisplatin</t> and melphalan sensitivity. (A and D) A549 cells were treated for 24 hours with the indicated concentration of cisplatin (A) or melphalan (D) in the presence of diluent (●) or A-443654 at 62.5 nM (▽)
    Cisplatin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10270 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore camptothecin
    Chk1‐dependent phosphorylation does not cause stabilization or subcellular relocalization of atypical E2Fs Protein expression of endogenous and exogenous atypical E2Fs in the HeLa/TO cell lines. EGFP‐tagged wild‐type, alanine and aspartic mouse constructs were incorporated in the HeLa/TO‐inducible system by single colony selection. Cells were then treated either without (Veh) or with (Dox) doxycycline for 24 h. Exogenous E2F7 was measured by anti‐GFP antibody, and endogenous E2F7 was measured by anti‐human E2F7 antibody. For E2F8, both exogenous and endogenous forms were detected by a single E2F8 antibody. Asterisks indicate specific detections from the antibodies. Protein expression of endogenous E2F7, E2F8, phos‐Chk1 S345 , and total Chk1 in U2OS cells treated with cycloheximide (CHX), in the presence or absence of etoposide. E2F7 and E2F8 proteins are localized in the nucleus. Inducible cell lines were treated with doxycycline and etoposide for 16 h, followed by isolation of cytosolic and nuclear protein fractions. HDAC1 expression was used as an indicator for enrichment of nuclear proteins. Immunofluorescence showing the localization of E2F7, E2F8, and MDC1 in response to <t>camptothecin</t> treatment for 8 h in U2OS cells. E2F7, E2F8, and MDC1 were labeled with GFP, and DNA damage sites are detected with an antibody against γ‐H2AX. DNA was stained with DAPI. Scale bar: 5 μm. Source data are available online for this figure.
    Camptothecin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3469 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore paclitaxel
    Deep (R)CNN model validation to predict toxicity and to extract knowledge for clustering of drugs based on their toxicity mechanisms. HL1 cells treated with one of 24 compounds or DMSO at the concentrations indicated (μM) (Experiments #11–14) were processed as described in the Materials and Methods. (A) Plots displaying mean toxicity readouts of four replicate wells, obtained from percentage of healthy cells predicted by the CNN Nuc (Tox_CNN) or RCNN (Tox_RCNN) mixed models, and from nuclei counting by standard image segmentation (Num Nuc), or by using RCNN-based automated detection (Num Nuc RCNN). For each well, toxicity readouts were obtained by computing Z-scores (normalizing to DMSO-treated wells) with adjustment of the sign to display toxic effects as positive values. (B) Hierarchical clustering of features obtained with the Tox_CNN model from HL1 cells treated with 25μM of the indicated drugs or 0.78μM <t>Taxol;</t> untreated (-); DMSO, control. Colors highlight mechanism of toxicity associated with compounds.
    Paclitaxel, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 5901 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore staurosporine
    Toxicity prediction using deep CNN strategies compared with established readouts. HL1 cells treated or not (-) with the indicated concentrations of compounds (μM) or DMSO were processed as described in the Materials and Methods. (A) Representative fluorescence microscopy images of DAPI-stained cells treated or not (Ctrl) with the highest concentrations of the indicated compounds in a reference experiment (Experiment #1) used for CNN training. (B-D) Boxplots of per-well toxicity assessments from established measurements: nucleus count (Num Nuc) (B), Caspase 3/7 nucleus:cytoplasm ratio (Casp nuc/cyto) (C), and Mitotracker cytoplasmic intensity (Mito) (D). (E) CNN architecture for predicting health status from single-cell image crops, as described in Materials and Methods. (F) Cropping strategies; representative image crops are shown of nucleus (Nuc), nucleus+cytoplasm (Cell), nucleus+margin (Nuc_Ring), and nucleus+cytoplasm+background+neighboring cells (All). (G) Boxplot of per-well toxicity assessment from CNN Nuc predictions (percentage of cells classified as healthy). (H) Plot displaying mean toxicity readouts of replicate wells, obtained from the percentage of healthy cells predicted by the different CNN models (CNN Nuc, Nuc_Ring, Cell, All, 4crops) and the standard nuclei counting (Num Nuc) for the different treatments indicated. For each well, toxicity readouts were obtained by computing Z-scores (normalizing to DMSO-treated wells) with adjustment of the sign to display toxic effects as positive values. Points and corresponding error bars represent the mean and standard error of the mean, respectively, of results obtained by evaluating the 5 different CNN models trained for each cropping strategy. (I) Evaluation performance of the different CNN models for predicting toxic effects of <t>staurosporine</t> assessed using Caspase 3/7 fluorescent reporter as reference, as described in the Materials and Methods. Boxplots display AUC values obtained with the 5 models trained for each cropping strategy. (J) Correlations between cell density and CNN predictions obtained with the different models for untreated cells. Boxplots represent Pearson correlation coefficient, R, obtained with all 5 models trained with each cropping strategy (top), and exemplary dotplots for each strategy (selecting the model with the median AUC among the five) including regression line, R value and significance (bottom). p-value: *
    Staurosporine, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 7861 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore mg132
    p300 is phosphorylated at S1834 upon UV irradiation by p38 MAPK and Akt. ( A ) OSU-2 cells were UV irradiated and further cultured for various time periods. p300 phosphorylation was detected using western blotting with anti-phospho-p300(S1834) or anti-phospho-p300(S89) antibodies. ( B ) OSU-2 cells were pretreated with <t>MG132</t> for 1 h, UV irradiated and further cultured for 8 h, the p300 and phospho-p300 proteins were detected as (A). ( C and D ) OSU-2 cells were pretreated with either p38 MAPK inhibitor SB203580 (C) or Akt inhibitor LY294002 (D) for 1 h, UV irradiated at 10 J/m 2 and further cultured in media containing inhibitors for various time periods. Whole cell lysates were prepared and subjected to western blotting to detect p300 and phospho-p300. pMK2 and pAkt were detected to show the inhibition of p38 MAPK and Akt activity, respectively. The intensity of phospho-p300(S1834) in each sample was quantified and normalized to Tubulin, the relative amount of phospho-p300(S1834) was calculated compared with control sample and plotted below each immunoblotting image.
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    99
    Millipore actinomycin d
    Damage-inducible Rad18–SHPRH binding coincides with loss of high molecular weight Rad18. (A) Inhibition of checkpoint kinases does not affect the Rad18–SHPRH interaction. Cells transfected with FLAG-Rad18 and GFP-SHPRH were mock treated or exposed to 0.005% MMS and the respective kinase inhibitors for 4 h before being lysed and analyzed as in Fig. 1 B . ATRi, ATR inhibition; ATMi, ATM inhibition. (B) Rad18 and SHPRH interact specifically after MMS or H 2 O 2 treatment. Cells expressing FLAG-Rad18 and GFP-SHPRH were treated with 50 ppm MMS, 50 J/m 2 UV, 30 µM mitomycin C (MMC), 0.1% ethyl methanesulfonate (EMS), 20 µM 4-NQO, 30 µM aphidicolin (Aph), 2 µM camptothecin (CPT), 1 µM <t>actinomycin</t> D (ActD), 20 µM etoposide (Etop), 0.1% hydrogen peroxide (H 2 O 2 ), or 50 µM cis-platinum for 4 h before being lysed under condition B and analyzed by Western blotting. (C) Endogenous Rad18 is deubiquitinated after MMS and H 2 O 2 treatment. Untransfected cells were treated with UV (0, 100, 200, and 400 J/m 2 ), MMS (0, 25, 50, and 100 ppm), or H 2 O 2 (0, 1, 2, and 4 mM) for 4 h before being lysed and analyzed by Western blotting.
    Actinomycin D, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12624 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore 5 fluorouracil
    mBMDC/67NR-Hyg clones exhibit an enhanced resistance towards several chemotherapeutic drugs . XTT proliferation assay was conducted to investigate whether the increased Abcb1a/Abcb1b expression levels of the mBMDC/67NR-Hyg clones were correlated with an enhanced drug resistance. mBMDC/67NR-Hyg clones showed a marked resistance towards 17-DMAG and doxorubicin as well as etoposide and paclitaxel, but not <t>5-Fluorouracil.</t> Inhibition of Abcb1a/Abcb1b activity by verapamil completely blocked the doxorubicin resistance of mBMDC/67NR-Hyg clones. By contrast, resistance of mBMDC/67NR-Hyg clones towards 17-DMAG, etoposide, and paclitaxel was partially impaired by verapamil indicating that additional ABC transporters or other drug resistance mechanisms are involved in the mBMDC/67NR-Hyg clones resistance towards these chemotherapeutic compounds.
    5 Fluorouracil, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3348 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore nocodazole
    Eusynstyelamide B (EB, 1 ) induces cell death in MDA-MB-231 cells through apoptosis. ( A ) MDA-MB-231 cells were treated with 2.5 µM EB or 0.1% DMSO for 96 h and imaged using an Olympus IX70 microscope (10× objective, scale bar = 100 µm); ( B ) Cells were treated with 2.5 µM EB for the indicated times or 0.1% DMSO for 96 h (C). As positive controls for apoptosis (PARP cleavage), cell were treated with 1 µM doxorubicin (Dox) for 48 h, 2 nM taxol (Tax) for 24 h or 83 nM <t>nocodazole</t> (Noc) for 24 h. As a positive control for autophagy (LC3B-II), cell were treated with 25 µM chloroquine (Cq) for 48 h. Expression of the indicated proteins was assessed by Western blotting, normalized against the level of β-actin, and is expressed in fold-change relative to control (C); ( C ) MDA-MB-231 cells were treated with 5 µM EB or 0.1% DMSO for 96 h, stained with PI and Annexin V-FITC, and the number of viable, early apoptotic and late apoptotic/necrotic cells was quantified by flow cytometry (mean ± SD, n = 3, * p
    Nocodazole, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 15733 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dmso
    Paclitaxel increases IRE1-dependent cytokine secretion. a MDA-MB-231 cells were treated with 10 nM paclitaxel for the indicated times, and cell lysates were immunoblotted for XBP1s and Actin. b MDA-MB-231 cells were treated with 10 nM paclitaxel in the presence of 20 μM MKC8866 or vehicle <t>(DMSO)</t> for 72 h, cell lysates were harvested and immunoblotted for XBP1s and Actin. c MDA-MB-231 cells were treated with 10 nM paclitaxel in combination with DMSO or 20 μM MKC8866 for 72 h in the presence of <t>Boc-D-fmk</t> (40 μM). Following treatment conditioned medium was collected and analyzed by ELISA for secretion of IL-6, IL-8, CXCL1, and GM-CSF. Cells were lysed and protein quantified ( n = 3). Results shown for a and b are representative of three independent experiments. * P
    Dmso, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 55308 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore nutlin 3
    Tumor suppressor p53 induces KLF17 expression. A , A549 cells were transfected with control siRNA or siRNA targeting p53 (20 n m ) for 48 h and then left untreated or treated with <t>Nutlin-3</t> (10 μ m ) for the indicated times. Total RNA was isolated and
    Nutlin 3, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 928 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore cycloheximide
    Tumor suppressor p53 induces KLF17 expression. A , A549 cells were transfected with control siRNA or siRNA targeting p53 (20 n m ) for 48 h and then left untreated or treated with <t>Nutlin-3</t> (10 μ m ) for the indicated times. Total RNA was isolated and
    Cycloheximide, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 18611 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore thapsigargin
    Alteration of BiP protein level in response to DNA damage. a HeLa cells were treated with the indicated concentration of <t>thapsigargin</t> (8 h) or etoposide (48 h), and a western blot analysis was performed using the indicated antibody. The
    Thapsigargin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 6594 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore hydrogen peroxide
    Alteration of BiP protein level in response to DNA damage. a HeLa cells were treated with the indicated concentration of <t>thapsigargin</t> (8 h) or etoposide (48 h), and a western blot analysis was performed using the indicated antibody. The
    Hydrogen Peroxide, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 16428 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore aphidicolin
    KAP1 phospho-Ser473 after DNA damage is Chk1- and Chk2-dependent . (a) Etoposide-induced KAP1 Ser473 phosphorylation is abolished by Chk1/Chk2 inhibition and reduced upon ATM inhibition. U2OS cells were untreated or treated with 5 μM etoposide (ETP) for 4 h in the presence or absence of KU55933 (ATMi), caffeine (Caff), or AZD7762 (AZD). (b) KAP1 phospho-Ser473 induction after 20 Gy of IR is abolished by AZD7762 (the drug was not removed during the recovery time). Chk2 phospho-Thr68 was used as readout of DNA damage and histone H3 phospho-Ser10 was used as readout for the G2/M checkpoint. (c) AZD7762 decreases KAP1 phospho-Ser473 on immunofluorescence; U2OS cells were treated as in (b). (d) KAP1 Ser473 is targeted by Chk1. U2OS cells were transfected with either siLuc, siChk1, siChk2, or both siChk1 and siChk2, then treated with 10 μM <t>aphidicolin</t> for 1 h. (e) KAP1 Ser473 is targeted by Chk2. U2OS cells were transfected as in (d) and treated as in (b). (f) KAP1 phospho-Ser473 is neither recruited nor excluded from laser-induced DNA-damage sites. Cells were fixed 5, 10 or 30 minutes after micro-irradiation.
    Aphidicolin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3944 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore propidium iodide
    Effects of HA-BAX on morphology and sub-G 0 content of SW626 cells treated with paclitaxel. Neo-control clone A10 and HA-BAX clone D7 were grown for 48 hr in the presence of medium alone or paclitaxel (0.05 μM), followed by morphologic assessment (Wrights–Giemsa staining) and quantitation of the sub-G 0 fraction by <t>propidium</t> iodide staining as described. Significant amounts of nuclear fragmentation and an increased fraction of sub-G 0 cells are observed for HA-BAX clone D7 compared with neo-control cells, consistent with paclitaxel-mediated apoptosis. Representative data from one of three separate experiments are shown.
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    Millipore dimethyl sulfoxide
    Effects of HA-BAX on morphology and sub-G 0 content of SW626 cells treated with paclitaxel. Neo-control clone A10 and HA-BAX clone D7 were grown for 48 hr in the presence of medium alone or paclitaxel (0.05 μM), followed by morphologic assessment (Wrights–Giemsa staining) and quantitation of the sub-G 0 fraction by <t>propidium</t> iodide staining as described. Significant amounts of nuclear fragmentation and an increased fraction of sub-G 0 cells are observed for HA-BAX clone D7 compared with neo-control cells, consistent with paclitaxel-mediated apoptosis. Representative data from one of three separate experiments are shown.
    Dimethyl Sulfoxide, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 27840 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rapamycin
    HMA induces autophagy. ( A ) ARPE 19 cells were untreated or treated with <t>rapamycin</t> for 2 hrs, with HMA or etoposide for 24 hrs and then probed with anti-LC3 antibody. Scale bar 25 μm. ( B ) ARPE-19 cells were treated as before and analysed by Western blot using anti-beclin 1, ATG-7, AGT5-12 and LC3 antibodies. γ tubulin was used as a loading control (left panel). On middle and right panels, cells were treated as before and analysed using anti-ERK and phospho-ERK (*) antibodies (middle panel) or with anti JNK1 or phosphorylated JNK1 (*) antibodies (right panel). Under the western images the quantification of the bands is reported showing a significant increase of Beclin 1 (Means are different from each other as calculated from a one-way anova test ( P
    Rapamycin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13301 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore tunicamycin
    Deletion of HtrA2/Omi results in increased sensitivity to mitochondrion-damaging agents. Thymocytes were isolated from control and HtrA2/Omi knockout animals and incubated with increasing concentrations of anti-Fas antibody (A), etoposide (B), or the mitochondrion-damaging agents CCCP (C) and rotenone (D). Viability was determined 16 h after treatment by flow cytometry using propidium iodide. Results are representative of three independent experiments. (E) Viability of simian virus 40 large-T-antigen-immortalized MEFs derived from wild-type (+/+) and HtrA2/Omi knockout animals, determined by measurement of sub-G 1 cell populations by flow cytometry. Cells were incubated in the presence of CCCP (25 μM for 27 h), rotenone (25 μM for 27 h), <t>tunicamycin</t> (2.5 μg/ml for 27 h), or hydrogen peroxide (3 μM for 4.5 h) and compared to untreated control cells. Results show the means ± standard deviations of results of three independent experiments. (F) The neuronalresponse to glutamate-induced cytotoxicity was determined in primary neurons isolated from E14.5 embryos cultured in vitro for 10 days. Following incubation with the indicated concentrations of glutamate for 4 h, cells were stained with Hoechst 33342 and nuclear morphology was determined. Results show the means ± standard deviations of results from two independent experiments where six fields with at least 40 cells were scored for each data point.
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    Millipore gemcitabine hydrochloride
    Functional analysis of the interaction between ANXA2 and p50 in Mia-Paca2 cells. All data are representative of three independent experiments. ( a ) Interaction between endogenous ANXA2 and p50 in Mia-Paca2 cells. Cell extracts were immunoprecipitated with anti-p50 rabbit polyclonal IgG or anti-rabbit preimmune IgG, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analyzed by western blotting with the indicated antibodies. ( b ) Mammalian expression vectors containing the indicated DNA sequences were introduced into Mia-Paca2 cells by electroporation, and a NF- κ B transcriptional activity assay was performed. ( c ) IL-6 secretion into the culture medium by cells expressing wild-type or Y23A ANXA2 assessed by an enzyme-linked immunosorbent assay. ( d ) Viability (WST reagent) of Mia-Paca2 cells expressing wild-type or Y23A ANXA2 after treatment with TNF- α for 48 h. ( e ) Viability (WST reagent) of Mia-Paca2 cells expressing wild-type or Y23A ANXA2 after treatment with <t>gemcitabine</t> treatment for 48 h. ( f ) Caspase 3 levels and poly ADP-ribose polymerase (PARP) cleavage were confirmed by western blotting analysis after gemcitabine treatment (5 μ M) for 48 h. ( g ) One day before gemcitabine treatment, Mia-Paca2 cells expressing wild-type or Y23A ANXA2 (7 × 10 5 cells) were plated into 100 mm cell culture plate. 1 μ M gemcitabine and 10 μ M Bay 11-7082 were treated for 36 h, and cleaved caspase 3 was confirmed by western blotting analysis. * P
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    Millipore bleomycin
    CREB3L1 activation is independent from DNA breaks. ( A ) On day 0, indicated cells were seeded at 4 × 10 5 cells per 60 mm dish. On day 1, cells were treated with 500 nM doxorubicin or 500 nM etoposide. On day 2, 24 hr after the treatment, the cells were harvested for immunoblot analysis with antibodies reacting against γH2AX or actin. ( B ) Huh7 cells were seeded and treated as described in ( A ). On day 2, cells were separated into nuclear and membrane fractions and analyzed by immunoblot analysis as described in Figure 1B . ( C ) The effect of etoposide on proliferation of the indicated cells was determined as described in Figure 3A . ( D )–( G ) On day 0, indicated cells were seeded at 1.5 × 10 5 cells per 60 mm dish. On day 1, cells were treated with indicated concentrations of etoposide ( D ), doxorubicin ( E ), <t>bleomycin</t> ( F ), or paclitaxel ( G ). On day 3, 48 hr after the treatment, proliferation of the cells was determined by measurement of cellular DNA. The amount of DNA just prior to the drug treatment and after treatment with no drug for 48 hr is set to 0% and 100%, respectively. ( C )–( G ) Results are reported as mean ± S.E.M. of three independent experiments. DOI: http://dx.doi.org/10.7554/eLife.00090.007
    Bleomycin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1572 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    JMY involved in <t>DNA</t> damage responses in maturing porcine oocyte. A : Typical staining of γ-H2AX in porcine oocytes before and after the treatment with <t>etoposide</t> (25 mg/mL) at MI (A) and MII (B) stage. Red, γ-H2AX; blue, chromatin. B : JMY expression in porcine oocytes on 44 h after etoposide. Note that JMY is localized at nucleus in etoposide treated group (indicated in the arrow). Red, JMY; blue, chromatin. Values represent mean ± s.e.m, * p
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    Pregnenolone sulphate-mediated insulin secretion is inhibited by <t>mefenamic</t> acid. INS-1E cells were cultured and insulin secretion measured as described in Methods . Test substances were added as indicated. (A) The effect of pregnenolone sulphate and mefenamic
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    ANK1 is upregulated following exposure to different inducers of DNA damage and in a variety of cell types. ( a ) Diagram showing the location of miR-486 in relation to the open reading frame of the cytoskeleton adaptor gene ANK1 , as well as the small ankyrin-1 isoform (sAnk1). MiR-486 is located in the intronic region between the last two exons of the ANK1 gene. ( b ) MCF10A cells were treated with doxorubicin (dox.) as in Figure 1c , and ANK1 mRNA levels were quantified by qPCR. The p53-regulated p21 (Waf1/ Cip1) transcript served as a positive control ( n =3). ( c ) Representative western analysis of protein samples harvested from cells undergoing DNA damage ( n =3). The ankyrin-1 protein (ANK1) (246 kDa) was quantified (right panel) and normalised to β -actin levels. ( d ) Ankyrin-1 was induced following ionising radiation (IR, 10 Gy) exposure in MCF10A cells but not by ultraviolet light-C (UVC, 100 J/m 2 ). Cells were harvested 24 h after IR/UVC exposure. Cisplatin (10 μ g/ml) and <t>mitomycin</t> C (300 nM) treatment for 24 h also induced ankyrin-1. Representative western blots are shown ( n =3). ( e ) Ankyrin-1 was upregulated following 25 μ M etoposide (etop.) treatment for 24 h in retinal pigment epithelium (RPE) and MCF7 cells, and in A549 cells following IR (5 Gy) exposure where cells were harvested after 6 h. Representative western blots are shown ( n =3). For all bar charts, values are mean±S.D. ( t -test, n =3, compared with 0 h control), * P
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    Image Search Results


    Validation of iTRAQ data through Western blot analysis. H2AX was selected for validation of the expression alteration trends through Western blot analysis. The results showed that H2AX is downregulated in Caki-1 cells compared with XIAP knockdown cells after etoposide treatment. GAPDH was used as a loading control. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; XIAP: X-linked inhibitor of apoptosis.

    Journal: Chinese Medical Journal

    Article Title: Isobaric tags for relative and absolute quantitation-based quantitative proteomic analysis of X-linked inhibitor of apoptosis and H2AX in etoposide-induced renal cell carcinoma apoptosis

    doi: 10.1097/CM9.0000000000000553

    Figure Lengend Snippet: Validation of iTRAQ data through Western blot analysis. H2AX was selected for validation of the expression alteration trends through Western blot analysis. The results showed that H2AX is downregulated in Caki-1 cells compared with XIAP knockdown cells after etoposide treatment. GAPDH was used as a loading control. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; XIAP: X-linked inhibitor of apoptosis.

    Article Snippet: At approximately 90% confluence, the cells were washed with phosphate-buffered saline and then treated with 60 μg/mL etoposide (Aldrich, Sigma, St. Louis, MO 63178, USA) or with serum-free medium as a control.

    Techniques: Western Blot, Expressing

    Interleukin 11 (IL-11)/IL-11R signaling pathway induced the chemo-resistance through JAK/STAT3 pathway. (A) Immunofluoresence of p-JAK2 in BGC823 and SGC7901 cells pre-treated with or without cultured medium of cancer-associated-fibroblasts (CAFs-CM)/rhIL-11 in the presence or absence of IL-11 neutralizing antibody. (B) Immunofluoresence of p-STAT3 in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (C) Western blotting of p-JAK2, total JAK2 and β-actin in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (D) Western blotting of p-STAT3, total STAT3, and β-actin in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (E) The cell viability of BGC823 cells treated with 6 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of ruxolitinib (5 μM). PBS, phosphate buffered saline; Dox, doxorubicin; Eto, etoposide. (F) The cell viability of SGC7901 cells treated with 4 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of ruxolitinib (5 μM). (G) The cell viability of BGC823 cells treated with 6 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the wild type or shSTAT3 cells. (H) The cell viability of SGC7901 cells treated with 4 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the wild type or shSTAT3 cells. The data was presented as the mean±standard error of mean from three independent experiments. ** p

    Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

    Article Title: Cancer-Associated Fibroblasts Promote the Chemo-resistance in Gastric Cancer through Secreting IL-11 Targeting JAK/STAT3/Bcl2 Pathway

    doi: 10.4143/crt.2018.031

    Figure Lengend Snippet: Interleukin 11 (IL-11)/IL-11R signaling pathway induced the chemo-resistance through JAK/STAT3 pathway. (A) Immunofluoresence of p-JAK2 in BGC823 and SGC7901 cells pre-treated with or without cultured medium of cancer-associated-fibroblasts (CAFs-CM)/rhIL-11 in the presence or absence of IL-11 neutralizing antibody. (B) Immunofluoresence of p-STAT3 in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (C) Western blotting of p-JAK2, total JAK2 and β-actin in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (D) Western blotting of p-STAT3, total STAT3, and β-actin in BGC823 and SGC7901 cells pre-treated with or without CAFs-CM/rhIL-11 (10 ng/mL) in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (E) The cell viability of BGC823 cells treated with 6 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of ruxolitinib (5 μM). PBS, phosphate buffered saline; Dox, doxorubicin; Eto, etoposide. (F) The cell viability of SGC7901 cells treated with 4 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of ruxolitinib (5 μM). (G) The cell viability of BGC823 cells treated with 6 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the wild type or shSTAT3 cells. (H) The cell viability of SGC7901 cells treated with 4 μg/mL DDP, 6 μM etoposide, and 6 μM doxorubicin respectively with or without CAFs-CM or rhIL-11 (10 ng/mL) pre-co-cultured in the wild type or shSTAT3 cells. The data was presented as the mean±standard error of mean from three independent experiments. ** p

    Article Snippet: Then BGC923 and SGC7901 were seeded, followed by adding drugs as following: cisplatin (DDP; 2, 4, 6, 8, 10, 12, and 16 μg/mL; Sigma), doxorubicin (1, 5, 25, 50, 200, 500, and 800 μM; Sigma) or etoposide (0.01, 0.1, 1, 2, 20, 200, and 500 μM; Sigma) for 48 hours.

    Techniques: Cell Culture, Western Blot

    Blockade interlukin (IL)-11/IL-11R signal relieves chemotherapy drug resistance in gastric cancer. (A) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells implants in phosphate buffered saline (PBS), doxorubicin, ruxolitinib, or doxorubicin combing ruxolitinib (left); the long-term survival of tumor bearing mice treated with PBS, doxorubicin, ruxolitinib, or doxorubicin combing ruxolitinib (right). Eto, etoposide; Dox, doxorubicin. (B) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells implants in PBS, etoposide, ruxolitinib, or etoposide combing ruxolitinib (left); the long-term survival of tumor bearing mice treated with PBS, etoposide, ruxolitinib, or etoposide combing ruxolitinib (right). (C) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells implants in PBS, DDP, ruxolitinib, or DDP combing ruxolitinib (left); the long-term survival of tumor bearing mice treated with PBS, DDP, ruxolitinib, or DDP combing ruxolitinib (right). (D) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 and then treated with PBS, doxorubicin, ruxolitinib, or doxorubicin combing ruxolitinib (left); the long-term survival of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 (2.5 μg/kg) and then treated with PBS, doxorubicin, ruxolitinib, or doxorubicin combing ruxolitinib (right). (E) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 (2.5 μg/kg) and then treated with PBS, etoposide, ruxolitinib, or etoposide combing ruxolitinib (left); the long-term survival of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 and then treated with PBS, etoposide, ruxolitinib, or etoposide combing ruxolitinib (right). (F) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 (2.5 μg/kg) and then treated with PBS, DDP, ruxolitinib, or DDP combing ruxolitinib (left); the long-term survival of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 and then treated with PBS, DDP, ruxolitinib, or DDP combing ruxolitinib (right). (G) The schematic diagram of drug resistance development induced by IL-11 in gastric cancer cells. The data was presented as the mean±standard error of mean from three independent experiments. ** p

    Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

    Article Title: Cancer-Associated Fibroblasts Promote the Chemo-resistance in Gastric Cancer through Secreting IL-11 Targeting JAK/STAT3/Bcl2 Pathway

    doi: 10.4143/crt.2018.031

    Figure Lengend Snippet: Blockade interlukin (IL)-11/IL-11R signal relieves chemotherapy drug resistance in gastric cancer. (A) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells implants in phosphate buffered saline (PBS), doxorubicin, ruxolitinib, or doxorubicin combing ruxolitinib (left); the long-term survival of tumor bearing mice treated with PBS, doxorubicin, ruxolitinib, or doxorubicin combing ruxolitinib (right). Eto, etoposide; Dox, doxorubicin. (B) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells implants in PBS, etoposide, ruxolitinib, or etoposide combing ruxolitinib (left); the long-term survival of tumor bearing mice treated with PBS, etoposide, ruxolitinib, or etoposide combing ruxolitinib (right). (C) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells implants in PBS, DDP, ruxolitinib, or DDP combing ruxolitinib (left); the long-term survival of tumor bearing mice treated with PBS, DDP, ruxolitinib, or DDP combing ruxolitinib (right). (D) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 and then treated with PBS, doxorubicin, ruxolitinib, or doxorubicin combing ruxolitinib (left); the long-term survival of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 (2.5 μg/kg) and then treated with PBS, doxorubicin, ruxolitinib, or doxorubicin combing ruxolitinib (right). (E) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 (2.5 μg/kg) and then treated with PBS, etoposide, ruxolitinib, or etoposide combing ruxolitinib (left); the long-term survival of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 and then treated with PBS, etoposide, ruxolitinib, or etoposide combing ruxolitinib (right). (F) The mean tumor volume of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 (2.5 μg/kg) and then treated with PBS, DDP, ruxolitinib, or DDP combing ruxolitinib (left); the long-term survival of NOD-SCID mice bearing SGC7901 cells injected with rhIL-11 and then treated with PBS, DDP, ruxolitinib, or DDP combing ruxolitinib (right). (G) The schematic diagram of drug resistance development induced by IL-11 in gastric cancer cells. The data was presented as the mean±standard error of mean from three independent experiments. ** p

    Article Snippet: Then BGC923 and SGC7901 were seeded, followed by adding drugs as following: cisplatin (DDP; 2, 4, 6, 8, 10, 12, and 16 μg/mL; Sigma), doxorubicin (1, 5, 25, 50, 200, 500, and 800 μM; Sigma) or etoposide (0.01, 0.1, 1, 2, 20, 200, and 500 μM; Sigma) for 48 hours.

    Techniques: Mouse Assay, Injection

    Interleukin 11 (IL-11) triggers the JAK/STAT3 pathway to elevate Bcl2 expression. (A) The expression of Bcl2 in BGC823 and SGC7901 cells treated with cultured medium of cancer-associated-fibroblasts (CAFs-CM) or rhIL-11 in the presence or absence of ruxolitinib (5 μM) in mRNA level. (B) The expression of Bcl2 in BGC823 and SGC7901 cells treated with CAFs-CM or rhIL-11 (10 ng/mL) in the presence or absence of ruxolitinib (5 μM) in protein level. (C) The cell viability of BGC823 cells treated with CAFs-CM or rhIL-11 in wild type or Bcl2 silenced cells. (D) The cell viability of SGC7901 cells treated with CAFs-CM or rhIL-11 in wild type or Bcl2 silenced cells. PBS, phosphate buffered saline; Dox, doxorubicin; Eto, etoposide. The data was presented as the mean±standard error of mean from three independent experiments. ** p

    Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

    Article Title: Cancer-Associated Fibroblasts Promote the Chemo-resistance in Gastric Cancer through Secreting IL-11 Targeting JAK/STAT3/Bcl2 Pathway

    doi: 10.4143/crt.2018.031

    Figure Lengend Snippet: Interleukin 11 (IL-11) triggers the JAK/STAT3 pathway to elevate Bcl2 expression. (A) The expression of Bcl2 in BGC823 and SGC7901 cells treated with cultured medium of cancer-associated-fibroblasts (CAFs-CM) or rhIL-11 in the presence or absence of ruxolitinib (5 μM) in mRNA level. (B) The expression of Bcl2 in BGC823 and SGC7901 cells treated with CAFs-CM or rhIL-11 (10 ng/mL) in the presence or absence of ruxolitinib (5 μM) in protein level. (C) The cell viability of BGC823 cells treated with CAFs-CM or rhIL-11 in wild type or Bcl2 silenced cells. (D) The cell viability of SGC7901 cells treated with CAFs-CM or rhIL-11 in wild type or Bcl2 silenced cells. PBS, phosphate buffered saline; Dox, doxorubicin; Eto, etoposide. The data was presented as the mean±standard error of mean from three independent experiments. ** p

    Article Snippet: Then BGC923 and SGC7901 were seeded, followed by adding drugs as following: cisplatin (DDP; 2, 4, 6, 8, 10, 12, and 16 μg/mL; Sigma), doxorubicin (1, 5, 25, 50, 200, 500, and 800 μM; Sigma) or etoposide (0.01, 0.1, 1, 2, 20, 200, and 500 μM; Sigma) for 48 hours.

    Techniques: Expressing, Cell Culture

    Cancer-associated-fibroblasts (CAFs) regulated chemo-resistance through secreting interleukin 11 (IL-11). The effect of CAFs on the sensitivity of gastric cancer cells to chemotherapy drugs was examined by using MTT assay. (A) The cell viability of BGC823 cells treated with 10 μg/mL DDP, 200 μM etoposide, and 20 μM doxorubicin, respectively with or without CAFs medium (CM) pretreated. Dox, doxorubicin; Eto, etoposide. (B) The cell viability of SGC7901 cells treated with 8 μg/mL DDP, 200 μM etoposide, and 20 μM doxorubicin. respectively with or without CM pretreated. (C) The mRNA expression of IL-11, SDF-1, HGF, FGF, PDGF, VEGF, and IL-1F9 in normal fibroblast and CAFs. (D) The expression of IL-11R in normal fibroblast and CAFs was detected by using enzyme-linked immunosorbent assay. (E) The mRNA expression of IL-11R, VEGFR, PDGFR, HGFR, CXCR4, SDF-1R, and IL-1F9R in SGC7901 cells with or without CAFs co-cultured. (F) The cell viability of BGC823 cells treated with 10 μg/mL DDP, 200 μM etoposide, and 20 μM doxorubicin, respectively with or without CAFs or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (G) The cell viability of SGC7901 cells treated with 8 μg/mL DDP, 200 μM etoposide, and 200 μM doxorubicin, respectively with or without CAFs or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (H) The tumor volume of NOD-SCID mice bearing SGC7901 cells co-injected with CAFs-CM (50 μL 10× CM) or IL-11 (2.5 μg/kg) treated by doxorubicin in the presence or absence of IL-11 neutralizing antibody (0.05 mg/kg). (I) The survival curve of NOD-SCID mice bearing SGC7901 cells co-injected with CAFsCM (50 μL 10× CM) or IL-11 treated by doxorubicin in the presence or absence of IL-11 neutralizing antibody (0.05 mg/kg). (J) Expression of IL-11 in gastric cancer tissues from chemo-sensitive and chemo-resistant patients. The data was presented as the mean±standard error of mean from three independent experiments. * p

    Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

    Article Title: Cancer-Associated Fibroblasts Promote the Chemo-resistance in Gastric Cancer through Secreting IL-11 Targeting JAK/STAT3/Bcl2 Pathway

    doi: 10.4143/crt.2018.031

    Figure Lengend Snippet: Cancer-associated-fibroblasts (CAFs) regulated chemo-resistance through secreting interleukin 11 (IL-11). The effect of CAFs on the sensitivity of gastric cancer cells to chemotherapy drugs was examined by using MTT assay. (A) The cell viability of BGC823 cells treated with 10 μg/mL DDP, 200 μM etoposide, and 20 μM doxorubicin, respectively with or without CAFs medium (CM) pretreated. Dox, doxorubicin; Eto, etoposide. (B) The cell viability of SGC7901 cells treated with 8 μg/mL DDP, 200 μM etoposide, and 20 μM doxorubicin. respectively with or without CM pretreated. (C) The mRNA expression of IL-11, SDF-1, HGF, FGF, PDGF, VEGF, and IL-1F9 in normal fibroblast and CAFs. (D) The expression of IL-11R in normal fibroblast and CAFs was detected by using enzyme-linked immunosorbent assay. (E) The mRNA expression of IL-11R, VEGFR, PDGFR, HGFR, CXCR4, SDF-1R, and IL-1F9R in SGC7901 cells with or without CAFs co-cultured. (F) The cell viability of BGC823 cells treated with 10 μg/mL DDP, 200 μM etoposide, and 20 μM doxorubicin, respectively with or without CAFs or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (G) The cell viability of SGC7901 cells treated with 8 μg/mL DDP, 200 μM etoposide, and 200 μM doxorubicin, respectively with or without CAFs or rhIL-11 (10 ng/mL) pre-co-cultured in the presence or absence of IL-11 neutralizing antibody (25 μg/mL). (H) The tumor volume of NOD-SCID mice bearing SGC7901 cells co-injected with CAFs-CM (50 μL 10× CM) or IL-11 (2.5 μg/kg) treated by doxorubicin in the presence or absence of IL-11 neutralizing antibody (0.05 mg/kg). (I) The survival curve of NOD-SCID mice bearing SGC7901 cells co-injected with CAFsCM (50 μL 10× CM) or IL-11 treated by doxorubicin in the presence or absence of IL-11 neutralizing antibody (0.05 mg/kg). (J) Expression of IL-11 in gastric cancer tissues from chemo-sensitive and chemo-resistant patients. The data was presented as the mean±standard error of mean from three independent experiments. * p

    Article Snippet: Then BGC923 and SGC7901 were seeded, followed by adding drugs as following: cisplatin (DDP; 2, 4, 6, 8, 10, 12, and 16 μg/mL; Sigma), doxorubicin (1, 5, 25, 50, 200, 500, and 800 μM; Sigma) or etoposide (0.01, 0.1, 1, 2, 20, 200, and 500 μM; Sigma) for 48 hours.

    Techniques: MTT Assay, Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Mouse Assay, Injection

    Enriched cancer-associated-fibroblasts (CAFs) enhanced the resistance of gastric cancer cells to chemotherapy. Accumulated CAFs in the chemotherapy-resistant gastric tumor sites and facilitate the resistance to chemotherapy drugs in gastric cancer cells. (A) The percentage of the cancer-associated fibroblasts in samples from chemo-sensitive (CS) and chemo-resistance (CR) gastric cancer patients was detected by flow cytometry. (B) The expression of the cancer-associated fibroblasts in samples from CS and CR gastric cancer patients was detected by immunohistochemistry. (C-H) The cell viability of SGC7901was detected after treated by different concentration of DDP (C), doxorubicin (D), and etoposide (E) pre-co-cultured with or without CAFs by using MTT assay. The cell viability of BGC823 was detected after treated by different concentration of DDP (F), doxorubicin (G), and etoposide (H) pre-co-cultured with or without CAFs by using MTT assay. The data was presented as mean±standard error of mean from three independent experiments. *** p

    Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

    Article Title: Cancer-Associated Fibroblasts Promote the Chemo-resistance in Gastric Cancer through Secreting IL-11 Targeting JAK/STAT3/Bcl2 Pathway

    doi: 10.4143/crt.2018.031

    Figure Lengend Snippet: Enriched cancer-associated-fibroblasts (CAFs) enhanced the resistance of gastric cancer cells to chemotherapy. Accumulated CAFs in the chemotherapy-resistant gastric tumor sites and facilitate the resistance to chemotherapy drugs in gastric cancer cells. (A) The percentage of the cancer-associated fibroblasts in samples from chemo-sensitive (CS) and chemo-resistance (CR) gastric cancer patients was detected by flow cytometry. (B) The expression of the cancer-associated fibroblasts in samples from CS and CR gastric cancer patients was detected by immunohistochemistry. (C-H) The cell viability of SGC7901was detected after treated by different concentration of DDP (C), doxorubicin (D), and etoposide (E) pre-co-cultured with or without CAFs by using MTT assay. The cell viability of BGC823 was detected after treated by different concentration of DDP (F), doxorubicin (G), and etoposide (H) pre-co-cultured with or without CAFs by using MTT assay. The data was presented as mean±standard error of mean from three independent experiments. *** p

    Article Snippet: Then BGC923 and SGC7901 were seeded, followed by adding drugs as following: cisplatin (DDP; 2, 4, 6, 8, 10, 12, and 16 μg/mL; Sigma), doxorubicin (1, 5, 25, 50, 200, 500, and 800 μM; Sigma) or etoposide (0.01, 0.1, 1, 2, 20, 200, and 500 μM; Sigma) for 48 hours.

    Techniques: Flow Cytometry, Cytometry, Expressing, Immunohistochemistry, Concentration Assay, Cell Culture, MTT Assay

    Synergistic effects of A-443654 on cisplatin and melphalan sensitivity. (A and D) A549 cells were treated for 24 hours with the indicated concentration of cisplatin (A) or melphalan (D) in the presence of diluent (●) or A-443654 at 62.5 nM (▽)

    Journal: Molecular Pharmacology

    Article Title: Context-Dependent Antagonism between Akt Inhibitors and Topoisomerase Poisons

    doi: 10.1124/mol.113.088674

    Figure Lengend Snippet: Synergistic effects of A-443654 on cisplatin and melphalan sensitivity. (A and D) A549 cells were treated for 24 hours with the indicated concentration of cisplatin (A) or melphalan (D) in the presence of diluent (●) or A-443654 at 62.5 nM (▽)

    Article Snippet: Additional reagents were purchased from the following suppliers: MK-2206 from ChemieTek (Indianapolis, IN); cisplatin, melphalan, etoposide, camptothecin, and propidium iodide (PI) from Sigma-Aldrich (St. Louis, MO); and Opti-MEM medium and Lipofectamine 2000 from Invitrogen (Carlsbad, CA).

    Techniques: Concentration Assay

    Chk1‐dependent phosphorylation does not cause stabilization or subcellular relocalization of atypical E2Fs Protein expression of endogenous and exogenous atypical E2Fs in the HeLa/TO cell lines. EGFP‐tagged wild‐type, alanine and aspartic mouse constructs were incorporated in the HeLa/TO‐inducible system by single colony selection. Cells were then treated either without (Veh) or with (Dox) doxycycline for 24 h. Exogenous E2F7 was measured by anti‐GFP antibody, and endogenous E2F7 was measured by anti‐human E2F7 antibody. For E2F8, both exogenous and endogenous forms were detected by a single E2F8 antibody. Asterisks indicate specific detections from the antibodies. Protein expression of endogenous E2F7, E2F8, phos‐Chk1 S345 , and total Chk1 in U2OS cells treated with cycloheximide (CHX), in the presence or absence of etoposide. E2F7 and E2F8 proteins are localized in the nucleus. Inducible cell lines were treated with doxycycline and etoposide for 16 h, followed by isolation of cytosolic and nuclear protein fractions. HDAC1 expression was used as an indicator for enrichment of nuclear proteins. Immunofluorescence showing the localization of E2F7, E2F8, and MDC1 in response to camptothecin treatment for 8 h in U2OS cells. E2F7, E2F8, and MDC1 were labeled with GFP, and DNA damage sites are detected with an antibody against γ‐H2AX. DNA was stained with DAPI. Scale bar: 5 μm. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Chk1 and 14‐3‐3 proteins inhibit atypical E2Fs to prevent a permanent cell cycle arrest

    doi: 10.15252/embj.201797877

    Figure Lengend Snippet: Chk1‐dependent phosphorylation does not cause stabilization or subcellular relocalization of atypical E2Fs Protein expression of endogenous and exogenous atypical E2Fs in the HeLa/TO cell lines. EGFP‐tagged wild‐type, alanine and aspartic mouse constructs were incorporated in the HeLa/TO‐inducible system by single colony selection. Cells were then treated either without (Veh) or with (Dox) doxycycline for 24 h. Exogenous E2F7 was measured by anti‐GFP antibody, and endogenous E2F7 was measured by anti‐human E2F7 antibody. For E2F8, both exogenous and endogenous forms were detected by a single E2F8 antibody. Asterisks indicate specific detections from the antibodies. Protein expression of endogenous E2F7, E2F8, phos‐Chk1 S345 , and total Chk1 in U2OS cells treated with cycloheximide (CHX), in the presence or absence of etoposide. E2F7 and E2F8 proteins are localized in the nucleus. Inducible cell lines were treated with doxycycline and etoposide for 16 h, followed by isolation of cytosolic and nuclear protein fractions. HDAC1 expression was used as an indicator for enrichment of nuclear proteins. Immunofluorescence showing the localization of E2F7, E2F8, and MDC1 in response to camptothecin treatment for 8 h in U2OS cells. E2F7, E2F8, and MDC1 were labeled with GFP, and DNA damage sites are detected with an antibody against γ‐H2AX. DNA was stained with DAPI. Scale bar: 5 μm. Source data are available online for this figure.

    Article Snippet: Other drugs used in this study were as follows: etoposide (10 μM, E1383, Sigma‐Aldrich); hydroxyurea (2 mM, H8627, Sigma‐Aldrich); thymidine (2 mM, T9250, Sigma‐Aldrich); camptothecin (20 μM, C9911, Sigma‐Aldrich), cycloheximide (50 μg/ml, 01810, Sigma‐Aldrich); UCN‐01(0.3 μM, U6508, Sigma‐Aldrich); and BV‐02 (5 nM, SML0140, Sigma‐Aldrich).

    Techniques: Expressing, Construct, Selection, Isolation, Immunofluorescence, Labeling, Staining

    Deep (R)CNN model validation to predict toxicity and to extract knowledge for clustering of drugs based on their toxicity mechanisms. HL1 cells treated with one of 24 compounds or DMSO at the concentrations indicated (μM) (Experiments #11–14) were processed as described in the Materials and Methods. (A) Plots displaying mean toxicity readouts of four replicate wells, obtained from percentage of healthy cells predicted by the CNN Nuc (Tox_CNN) or RCNN (Tox_RCNN) mixed models, and from nuclei counting by standard image segmentation (Num Nuc), or by using RCNN-based automated detection (Num Nuc RCNN). For each well, toxicity readouts were obtained by computing Z-scores (normalizing to DMSO-treated wells) with adjustment of the sign to display toxic effects as positive values. (B) Hierarchical clustering of features obtained with the Tox_CNN model from HL1 cells treated with 25μM of the indicated drugs or 0.78μM Taxol; untreated (-); DMSO, control. Colors highlight mechanism of toxicity associated with compounds.

    Journal: PLoS Computational Biology

    Article Title: Tox_(R)CNN: Deep learning-based nuclei profiling tool for drug toxicity screening

    doi: 10.1371/journal.pcbi.1006238

    Figure Lengend Snippet: Deep (R)CNN model validation to predict toxicity and to extract knowledge for clustering of drugs based on their toxicity mechanisms. HL1 cells treated with one of 24 compounds or DMSO at the concentrations indicated (μM) (Experiments #11–14) were processed as described in the Materials and Methods. (A) Plots displaying mean toxicity readouts of four replicate wells, obtained from percentage of healthy cells predicted by the CNN Nuc (Tox_CNN) or RCNN (Tox_RCNN) mixed models, and from nuclei counting by standard image segmentation (Num Nuc), or by using RCNN-based automated detection (Num Nuc RCNN). For each well, toxicity readouts were obtained by computing Z-scores (normalizing to DMSO-treated wells) with adjustment of the sign to display toxic effects as positive values. (B) Hierarchical clustering of features obtained with the Tox_CNN model from HL1 cells treated with 25μM of the indicated drugs or 0.78μM Taxol; untreated (-); DMSO, control. Colors highlight mechanism of toxicity associated with compounds.

    Article Snippet: Dimethyl sulfoxide vehicle (DMSO); Acetaminophen (ACETA); Doxorubicin (DOXO); carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP); Sunitinib; Staurosporine (STAUR); Paclitaxel (TAXOL); Imatinib; Thapsigargin; Gemcitabine; Quercetin; Atenolol; Simvastatin; Genistein; Vinblastine; Monensin; Anagrelide; Epirubicin; Etoposide and Lovastatin were from Sigma.

    Techniques:

    Toxicity prediction using deep CNN strategies compared with established readouts. HL1 cells treated or not (-) with the indicated concentrations of compounds (μM) or DMSO were processed as described in the Materials and Methods. (A) Representative fluorescence microscopy images of DAPI-stained cells treated or not (Ctrl) with the highest concentrations of the indicated compounds in a reference experiment (Experiment #1) used for CNN training. (B-D) Boxplots of per-well toxicity assessments from established measurements: nucleus count (Num Nuc) (B), Caspase 3/7 nucleus:cytoplasm ratio (Casp nuc/cyto) (C), and Mitotracker cytoplasmic intensity (Mito) (D). (E) CNN architecture for predicting health status from single-cell image crops, as described in Materials and Methods. (F) Cropping strategies; representative image crops are shown of nucleus (Nuc), nucleus+cytoplasm (Cell), nucleus+margin (Nuc_Ring), and nucleus+cytoplasm+background+neighboring cells (All). (G) Boxplot of per-well toxicity assessment from CNN Nuc predictions (percentage of cells classified as healthy). (H) Plot displaying mean toxicity readouts of replicate wells, obtained from the percentage of healthy cells predicted by the different CNN models (CNN Nuc, Nuc_Ring, Cell, All, 4crops) and the standard nuclei counting (Num Nuc) for the different treatments indicated. For each well, toxicity readouts were obtained by computing Z-scores (normalizing to DMSO-treated wells) with adjustment of the sign to display toxic effects as positive values. Points and corresponding error bars represent the mean and standard error of the mean, respectively, of results obtained by evaluating the 5 different CNN models trained for each cropping strategy. (I) Evaluation performance of the different CNN models for predicting toxic effects of staurosporine assessed using Caspase 3/7 fluorescent reporter as reference, as described in the Materials and Methods. Boxplots display AUC values obtained with the 5 models trained for each cropping strategy. (J) Correlations between cell density and CNN predictions obtained with the different models for untreated cells. Boxplots represent Pearson correlation coefficient, R, obtained with all 5 models trained with each cropping strategy (top), and exemplary dotplots for each strategy (selecting the model with the median AUC among the five) including regression line, R value and significance (bottom). p-value: *

    Journal: PLoS Computational Biology

    Article Title: Tox_(R)CNN: Deep learning-based nuclei profiling tool for drug toxicity screening

    doi: 10.1371/journal.pcbi.1006238

    Figure Lengend Snippet: Toxicity prediction using deep CNN strategies compared with established readouts. HL1 cells treated or not (-) with the indicated concentrations of compounds (μM) or DMSO were processed as described in the Materials and Methods. (A) Representative fluorescence microscopy images of DAPI-stained cells treated or not (Ctrl) with the highest concentrations of the indicated compounds in a reference experiment (Experiment #1) used for CNN training. (B-D) Boxplots of per-well toxicity assessments from established measurements: nucleus count (Num Nuc) (B), Caspase 3/7 nucleus:cytoplasm ratio (Casp nuc/cyto) (C), and Mitotracker cytoplasmic intensity (Mito) (D). (E) CNN architecture for predicting health status from single-cell image crops, as described in Materials and Methods. (F) Cropping strategies; representative image crops are shown of nucleus (Nuc), nucleus+cytoplasm (Cell), nucleus+margin (Nuc_Ring), and nucleus+cytoplasm+background+neighboring cells (All). (G) Boxplot of per-well toxicity assessment from CNN Nuc predictions (percentage of cells classified as healthy). (H) Plot displaying mean toxicity readouts of replicate wells, obtained from the percentage of healthy cells predicted by the different CNN models (CNN Nuc, Nuc_Ring, Cell, All, 4crops) and the standard nuclei counting (Num Nuc) for the different treatments indicated. For each well, toxicity readouts were obtained by computing Z-scores (normalizing to DMSO-treated wells) with adjustment of the sign to display toxic effects as positive values. Points and corresponding error bars represent the mean and standard error of the mean, respectively, of results obtained by evaluating the 5 different CNN models trained for each cropping strategy. (I) Evaluation performance of the different CNN models for predicting toxic effects of staurosporine assessed using Caspase 3/7 fluorescent reporter as reference, as described in the Materials and Methods. Boxplots display AUC values obtained with the 5 models trained for each cropping strategy. (J) Correlations between cell density and CNN predictions obtained with the different models for untreated cells. Boxplots represent Pearson correlation coefficient, R, obtained with all 5 models trained with each cropping strategy (top), and exemplary dotplots for each strategy (selecting the model with the median AUC among the five) including regression line, R value and significance (bottom). p-value: *

    Article Snippet: Dimethyl sulfoxide vehicle (DMSO); Acetaminophen (ACETA); Doxorubicin (DOXO); carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP); Sunitinib; Staurosporine (STAUR); Paclitaxel (TAXOL); Imatinib; Thapsigargin; Gemcitabine; Quercetin; Atenolol; Simvastatin; Genistein; Vinblastine; Monensin; Anagrelide; Epirubicin; Etoposide and Lovastatin were from Sigma.

    Techniques: Fluorescence, Microscopy, Staining

    p300 is phosphorylated at S1834 upon UV irradiation by p38 MAPK and Akt. ( A ) OSU-2 cells were UV irradiated and further cultured for various time periods. p300 phosphorylation was detected using western blotting with anti-phospho-p300(S1834) or anti-phospho-p300(S89) antibodies. ( B ) OSU-2 cells were pretreated with MG132 for 1 h, UV irradiated and further cultured for 8 h, the p300 and phospho-p300 proteins were detected as (A). ( C and D ) OSU-2 cells were pretreated with either p38 MAPK inhibitor SB203580 (C) or Akt inhibitor LY294002 (D) for 1 h, UV irradiated at 10 J/m 2 and further cultured in media containing inhibitors for various time periods. Whole cell lysates were prepared and subjected to western blotting to detect p300 and phospho-p300. pMK2 and pAkt were detected to show the inhibition of p38 MAPK and Akt activity, respectively. The intensity of phospho-p300(S1834) in each sample was quantified and normalized to Tubulin, the relative amount of phospho-p300(S1834) was calculated compared with control sample and plotted below each immunoblotting image.

    Journal: Nucleic Acids Research

    Article Title: p38 MAPK- and Akt-mediated p300 phosphorylation regulates its degradation to facilitate nucleotide excision repair

    doi: 10.1093/nar/gks1312

    Figure Lengend Snippet: p300 is phosphorylated at S1834 upon UV irradiation by p38 MAPK and Akt. ( A ) OSU-2 cells were UV irradiated and further cultured for various time periods. p300 phosphorylation was detected using western blotting with anti-phospho-p300(S1834) or anti-phospho-p300(S89) antibodies. ( B ) OSU-2 cells were pretreated with MG132 for 1 h, UV irradiated and further cultured for 8 h, the p300 and phospho-p300 proteins were detected as (A). ( C and D ) OSU-2 cells were pretreated with either p38 MAPK inhibitor SB203580 (C) or Akt inhibitor LY294002 (D) for 1 h, UV irradiated at 10 J/m 2 and further cultured in media containing inhibitors for various time periods. Whole cell lysates were prepared and subjected to western blotting to detect p300 and phospho-p300. pMK2 and pAkt were detected to show the inhibition of p38 MAPK and Akt activity, respectively. The intensity of phospho-p300(S1834) in each sample was quantified and normalized to Tubulin, the relative amount of phospho-p300(S1834) was calculated compared with control sample and plotted below each immunoblotting image.

    Article Snippet: To inhibit the activity of 26S proteasome, p38 MAPK or Akt, cells were first cultured in medium containing MG132 (Calbiochem, Billerica, MA, USA), SB203580 or LY294002 (Cell signaling Technology, Danvers, MA, USA) for 1 h, then treated with UV and further cultured in the medium containing inhibitors for various time periods.

    Techniques: Irradiation, Cell Culture, Western Blot, Inhibition, Activity Assay

    p300 is degraded upon UV or cisplatin, but not IR or Etoposide, treatments. (A and B) OSU-2 cells were exposed to UV, cisplatin ( A ), IR or Etoposide ( B ). The p300 level was detected by using western blotting. ( C ) OSU-2 cells were pre-treated with MG132 for 1 h, UV irradiated and the p300 protein was detected at the desired time point.

    Journal: Nucleic Acids Research

    Article Title: p38 MAPK- and Akt-mediated p300 phosphorylation regulates its degradation to facilitate nucleotide excision repair

    doi: 10.1093/nar/gks1312

    Figure Lengend Snippet: p300 is degraded upon UV or cisplatin, but not IR or Etoposide, treatments. (A and B) OSU-2 cells were exposed to UV, cisplatin ( A ), IR or Etoposide ( B ). The p300 level was detected by using western blotting. ( C ) OSU-2 cells were pre-treated with MG132 for 1 h, UV irradiated and the p300 protein was detected at the desired time point.

    Article Snippet: To inhibit the activity of 26S proteasome, p38 MAPK or Akt, cells were first cultured in medium containing MG132 (Calbiochem, Billerica, MA, USA), SB203580 or LY294002 (Cell signaling Technology, Danvers, MA, USA) for 1 h, then treated with UV and further cultured in the medium containing inhibitors for various time periods.

    Techniques: Western Blot, Irradiation

    Damage-inducible Rad18–SHPRH binding coincides with loss of high molecular weight Rad18. (A) Inhibition of checkpoint kinases does not affect the Rad18–SHPRH interaction. Cells transfected with FLAG-Rad18 and GFP-SHPRH were mock treated or exposed to 0.005% MMS and the respective kinase inhibitors for 4 h before being lysed and analyzed as in Fig. 1 B . ATRi, ATR inhibition; ATMi, ATM inhibition. (B) Rad18 and SHPRH interact specifically after MMS or H 2 O 2 treatment. Cells expressing FLAG-Rad18 and GFP-SHPRH were treated with 50 ppm MMS, 50 J/m 2 UV, 30 µM mitomycin C (MMC), 0.1% ethyl methanesulfonate (EMS), 20 µM 4-NQO, 30 µM aphidicolin (Aph), 2 µM camptothecin (CPT), 1 µM actinomycin D (ActD), 20 µM etoposide (Etop), 0.1% hydrogen peroxide (H 2 O 2 ), or 50 µM cis-platinum for 4 h before being lysed under condition B and analyzed by Western blotting. (C) Endogenous Rad18 is deubiquitinated after MMS and H 2 O 2 treatment. Untransfected cells were treated with UV (0, 100, 200, and 400 J/m 2 ), MMS (0, 25, 50, and 100 ppm), or H 2 O 2 (0, 1, 2, and 4 mM) for 4 h before being lysed and analyzed by Western blotting.

    Journal: The Journal of Cell Biology

    Article Title: DNA damage-specific deubiquitination regulates Rad18 functions to suppress mutagenesis

    doi: 10.1083/jcb.201311063

    Figure Lengend Snippet: Damage-inducible Rad18–SHPRH binding coincides with loss of high molecular weight Rad18. (A) Inhibition of checkpoint kinases does not affect the Rad18–SHPRH interaction. Cells transfected with FLAG-Rad18 and GFP-SHPRH were mock treated or exposed to 0.005% MMS and the respective kinase inhibitors for 4 h before being lysed and analyzed as in Fig. 1 B . ATRi, ATR inhibition; ATMi, ATM inhibition. (B) Rad18 and SHPRH interact specifically after MMS or H 2 O 2 treatment. Cells expressing FLAG-Rad18 and GFP-SHPRH were treated with 50 ppm MMS, 50 J/m 2 UV, 30 µM mitomycin C (MMC), 0.1% ethyl methanesulfonate (EMS), 20 µM 4-NQO, 30 µM aphidicolin (Aph), 2 µM camptothecin (CPT), 1 µM actinomycin D (ActD), 20 µM etoposide (Etop), 0.1% hydrogen peroxide (H 2 O 2 ), or 50 µM cis-platinum for 4 h before being lysed under condition B and analyzed by Western blotting. (C) Endogenous Rad18 is deubiquitinated after MMS and H 2 O 2 treatment. Untransfected cells were treated with UV (0, 100, 200, and 400 J/m 2 ), MMS (0, 25, 50, and 100 ppm), or H 2 O 2 (0, 1, 2, and 4 mM) for 4 h before being lysed and analyzed by Western blotting.

    Article Snippet: Cells were damaged with MMS (Sigma-Aldrich), mitomycin C (Sigma-Aldrich), EMS (Sigma-Aldrich), 4-nitroquinoline 1-oxide (Sigma-Aldrich), aphidicolin (Sigma-Aldrich), camptothecin (Sigma-Aldrich), actinomycin D (EMD Millipore), etoposide (Sigma-Aldrich), hydrogen peroxide (Sigma-Aldrich), or cis-platinum (Sigma-Aldrich) at the indicated concentrations and times.

    Techniques: Binding Assay, Molecular Weight, Inhibition, Transfection, Expressing, Cycling Probe Technology, Western Blot

    mBMDC/67NR-Hyg clones exhibit an enhanced resistance towards several chemotherapeutic drugs . XTT proliferation assay was conducted to investigate whether the increased Abcb1a/Abcb1b expression levels of the mBMDC/67NR-Hyg clones were correlated with an enhanced drug resistance. mBMDC/67NR-Hyg clones showed a marked resistance towards 17-DMAG and doxorubicin as well as etoposide and paclitaxel, but not 5-Fluorouracil. Inhibition of Abcb1a/Abcb1b activity by verapamil completely blocked the doxorubicin resistance of mBMDC/67NR-Hyg clones. By contrast, resistance of mBMDC/67NR-Hyg clones towards 17-DMAG, etoposide, and paclitaxel was partially impaired by verapamil indicating that additional ABC transporters or other drug resistance mechanisms are involved in the mBMDC/67NR-Hyg clones resistance towards these chemotherapeutic compounds.

    Journal: Cancer Cell International

    Article Title: Co-cultivation of murine BMDCs with 67NR mouse mammary carcinoma cells give rise to highly drug resistant cells

    doi: 10.1186/1475-2867-11-21

    Figure Lengend Snippet: mBMDC/67NR-Hyg clones exhibit an enhanced resistance towards several chemotherapeutic drugs . XTT proliferation assay was conducted to investigate whether the increased Abcb1a/Abcb1b expression levels of the mBMDC/67NR-Hyg clones were correlated with an enhanced drug resistance. mBMDC/67NR-Hyg clones showed a marked resistance towards 17-DMAG and doxorubicin as well as etoposide and paclitaxel, but not 5-Fluorouracil. Inhibition of Abcb1a/Abcb1b activity by verapamil completely blocked the doxorubicin resistance of mBMDC/67NR-Hyg clones. By contrast, resistance of mBMDC/67NR-Hyg clones towards 17-DMAG, etoposide, and paclitaxel was partially impaired by verapamil indicating that additional ABC transporters or other drug resistance mechanisms are involved in the mBMDC/67NR-Hyg clones resistance towards these chemotherapeutic compounds.

    Article Snippet: After 24 h media was replaced by culture media containing different concentrations of either 5-Fluorouracil (5-FU), 17-DMAG, doxorubicin, etoposide or paclitaxel (all drugs were purchased from Sigma Aldrich, Taufkirchen, Germany).

    Techniques: Clone Assay, Proliferation Assay, Expressing, Inhibition, Activity Assay

    Eusynstyelamide B (EB, 1 ) induces cell death in MDA-MB-231 cells through apoptosis. ( A ) MDA-MB-231 cells were treated with 2.5 µM EB or 0.1% DMSO for 96 h and imaged using an Olympus IX70 microscope (10× objective, scale bar = 100 µm); ( B ) Cells were treated with 2.5 µM EB for the indicated times or 0.1% DMSO for 96 h (C). As positive controls for apoptosis (PARP cleavage), cell were treated with 1 µM doxorubicin (Dox) for 48 h, 2 nM taxol (Tax) for 24 h or 83 nM nocodazole (Noc) for 24 h. As a positive control for autophagy (LC3B-II), cell were treated with 25 µM chloroquine (Cq) for 48 h. Expression of the indicated proteins was assessed by Western blotting, normalized against the level of β-actin, and is expressed in fold-change relative to control (C); ( C ) MDA-MB-231 cells were treated with 5 µM EB or 0.1% DMSO for 96 h, stained with PI and Annexin V-FITC, and the number of viable, early apoptotic and late apoptotic/necrotic cells was quantified by flow cytometry (mean ± SD, n = 3, * p

    Journal: Marine Drugs

    Article Title: Identification of Eusynstyelamide B as a Potent Cell Cycle Inhibitor Following the Generation and Screening of an Ascidian-Derived Extract Library Using a Real Time Cell Analyzer

    doi: 10.3390/md12105222

    Figure Lengend Snippet: Eusynstyelamide B (EB, 1 ) induces cell death in MDA-MB-231 cells through apoptosis. ( A ) MDA-MB-231 cells were treated with 2.5 µM EB or 0.1% DMSO for 96 h and imaged using an Olympus IX70 microscope (10× objective, scale bar = 100 µm); ( B ) Cells were treated with 2.5 µM EB for the indicated times or 0.1% DMSO for 96 h (C). As positive controls for apoptosis (PARP cleavage), cell were treated with 1 µM doxorubicin (Dox) for 48 h, 2 nM taxol (Tax) for 24 h or 83 nM nocodazole (Noc) for 24 h. As a positive control for autophagy (LC3B-II), cell were treated with 25 µM chloroquine (Cq) for 48 h. Expression of the indicated proteins was assessed by Western blotting, normalized against the level of β-actin, and is expressed in fold-change relative to control (C); ( C ) MDA-MB-231 cells were treated with 5 µM EB or 0.1% DMSO for 96 h, stained with PI and Annexin V-FITC, and the number of viable, early apoptotic and late apoptotic/necrotic cells was quantified by flow cytometry (mean ± SD, n = 3, * p

    Article Snippet: As positive controls, cells were treated with doxorubicin (1 µM, 48 h), etoposide (25 µM, 24 h), chloroquine (25 µM, 48 h), taxol (2 nM, 24 h), or nocodazole (83 nM, 24 h) which were purchased from Sigma Aldrich (Sigma-Aldrich, St. Louis, MO, USA).

    Techniques: Multiple Displacement Amplification, Microscopy, Positive Control, Expressing, Western Blot, Staining, Flow Cytometry, Cytometry

    Paclitaxel increases IRE1-dependent cytokine secretion. a MDA-MB-231 cells were treated with 10 nM paclitaxel for the indicated times, and cell lysates were immunoblotted for XBP1s and Actin. b MDA-MB-231 cells were treated with 10 nM paclitaxel in the presence of 20 μM MKC8866 or vehicle (DMSO) for 72 h, cell lysates were harvested and immunoblotted for XBP1s and Actin. c MDA-MB-231 cells were treated with 10 nM paclitaxel in combination with DMSO or 20 μM MKC8866 for 72 h in the presence of Boc-D-fmk (40 μM). Following treatment conditioned medium was collected and analyzed by ELISA for secretion of IL-6, IL-8, CXCL1, and GM-CSF. Cells were lysed and protein quantified ( n = 3). Results shown for a and b are representative of three independent experiments. * P

    Journal: Nature Communications

    Article Title: Inhibition of IRE1 RNase activity modulates the tumor cell secretome and enhances response to chemotherapy

    doi: 10.1038/s41467-018-05763-8

    Figure Lengend Snippet: Paclitaxel increases IRE1-dependent cytokine secretion. a MDA-MB-231 cells were treated with 10 nM paclitaxel for the indicated times, and cell lysates were immunoblotted for XBP1s and Actin. b MDA-MB-231 cells were treated with 10 nM paclitaxel in the presence of 20 μM MKC8866 or vehicle (DMSO) for 72 h, cell lysates were harvested and immunoblotted for XBP1s and Actin. c MDA-MB-231 cells were treated with 10 nM paclitaxel in combination with DMSO or 20 μM MKC8866 for 72 h in the presence of Boc-D-fmk (40 μM). Following treatment conditioned medium was collected and analyzed by ELISA for secretion of IL-6, IL-8, CXCL1, and GM-CSF. Cells were lysed and protein quantified ( n = 3). Results shown for a and b are representative of three independent experiments. * P

    Article Snippet: Cells were treated with the indicated concentrations of MKC8866 (Fosun Orinove PharmaTech Inc.), Tunicamycin (Tm) (Sigma-Aldrich, T7765), Thapsigargin (Tg) (Sigma-Aldrich, T9033), Paclitaxel (Sigma-Aldrich, T7402), Boc-D-fmk (Biovision, 1160-5), Etoposide (Sigma-Aldrich, E1383), or an equal volume of DMSO (Sigma-Aldrich, D2650).

    Techniques: Multiple Displacement Amplification, Enzyme-linked Immunosorbent Assay

    Tumor suppressor p53 induces KLF17 expression. A , A549 cells were transfected with control siRNA or siRNA targeting p53 (20 n m ) for 48 h and then left untreated or treated with Nutlin-3 (10 μ m ) for the indicated times. Total RNA was isolated and

    Journal: The Journal of Biological Chemistry

    Article Title: Tumor-suppressive p53 Signaling Empowers Metastatic Inhibitor KLF17-dependent Transcription to Overcome Tumorigenesis in Non-small Cell Lung Cancer *

    doi: 10.1074/jbc.M114.635730

    Figure Lengend Snippet: Tumor suppressor p53 induces KLF17 expression. A , A549 cells were transfected with control siRNA or siRNA targeting p53 (20 n m ) for 48 h and then left untreated or treated with Nutlin-3 (10 μ m ) for the indicated times. Total RNA was isolated and

    Article Snippet: For cell treatments, we used Nutlin-3 (10 μ m ) (Sigma- Aldrich), Adriamycin (0.5 μ m ) (Sigma-Aldrich), and etoposide (10 μ m ) (Sigma-Aldrich).

    Techniques: Expressing, Transfection, Isolation

    p53 interacts with and recruits p300 to KLF17 promoter in response to chemotherapy. A , schematic representation of ChIP primers from the KLF17 promoter. B and C , A549 cells were left untreated or treated with Nutlin-3 and Adriamycin for 24 h, and chromatin

    Journal: The Journal of Biological Chemistry

    Article Title: Tumor-suppressive p53 Signaling Empowers Metastatic Inhibitor KLF17-dependent Transcription to Overcome Tumorigenesis in Non-small Cell Lung Cancer *

    doi: 10.1074/jbc.M114.635730

    Figure Lengend Snippet: p53 interacts with and recruits p300 to KLF17 promoter in response to chemotherapy. A , schematic representation of ChIP primers from the KLF17 promoter. B and C , A549 cells were left untreated or treated with Nutlin-3 and Adriamycin for 24 h, and chromatin

    Article Snippet: For cell treatments, we used Nutlin-3 (10 μ m ) (Sigma- Aldrich), Adriamycin (0.5 μ m ) (Sigma-Aldrich), and etoposide (10 μ m ) (Sigma-Aldrich).

    Techniques: Chromatin Immunoprecipitation

    Depletion of KLF17 affects the cytostatic function of p53. A and B , A549 cells was transfected with control siRNA or siRNA targeting KLF17 (20 n m ) for 48 h and then treated with Nutlin-3 (10 μ m ) for 24 h. Total RNA was extracted and subjected

    Journal: The Journal of Biological Chemistry

    Article Title: Tumor-suppressive p53 Signaling Empowers Metastatic Inhibitor KLF17-dependent Transcription to Overcome Tumorigenesis in Non-small Cell Lung Cancer *

    doi: 10.1074/jbc.M114.635730

    Figure Lengend Snippet: Depletion of KLF17 affects the cytostatic function of p53. A and B , A549 cells was transfected with control siRNA or siRNA targeting KLF17 (20 n m ) for 48 h and then treated with Nutlin-3 (10 μ m ) for 24 h. Total RNA was extracted and subjected

    Article Snippet: For cell treatments, we used Nutlin-3 (10 μ m ) (Sigma- Aldrich), Adriamycin (0.5 μ m ) (Sigma-Aldrich), and etoposide (10 μ m ) (Sigma-Aldrich).

    Techniques: Transfection

    Overexpression of KLF17 enhances p53 tumor-suppressive function. A , A549 cells were transfected with FLAG-vector or vector encoding KLF17 plasmid for 24 h and then left untreated or treated with Nutlin-3 (10 μ m ) for 24 h, and an MTT assay was

    Journal: The Journal of Biological Chemistry

    Article Title: Tumor-suppressive p53 Signaling Empowers Metastatic Inhibitor KLF17-dependent Transcription to Overcome Tumorigenesis in Non-small Cell Lung Cancer *

    doi: 10.1074/jbc.M114.635730

    Figure Lengend Snippet: Overexpression of KLF17 enhances p53 tumor-suppressive function. A , A549 cells were transfected with FLAG-vector or vector encoding KLF17 plasmid for 24 h and then left untreated or treated with Nutlin-3 (10 μ m ) for 24 h, and an MTT assay was

    Article Snippet: For cell treatments, we used Nutlin-3 (10 μ m ) (Sigma- Aldrich), Adriamycin (0.5 μ m ) (Sigma-Aldrich), and etoposide (10 μ m ) (Sigma-Aldrich).

    Techniques: Over Expression, Transfection, Plasmid Preparation, MTT Assay

    Nutlin-3 recruits KLF17 to EMT gene promoters in a p53-dependent manner. A , A549 cells were left untreated or treated with Nutlin-3 (10 μ m ) for 24 h, and chromatin immunoprecipitation was performed with the indicated antibodies. B–D , A549

    Journal: The Journal of Biological Chemistry

    Article Title: Tumor-suppressive p53 Signaling Empowers Metastatic Inhibitor KLF17-dependent Transcription to Overcome Tumorigenesis in Non-small Cell Lung Cancer *

    doi: 10.1074/jbc.M114.635730

    Figure Lengend Snippet: Nutlin-3 recruits KLF17 to EMT gene promoters in a p53-dependent manner. A , A549 cells were left untreated or treated with Nutlin-3 (10 μ m ) for 24 h, and chromatin immunoprecipitation was performed with the indicated antibodies. B–D , A549

    Article Snippet: For cell treatments, we used Nutlin-3 (10 μ m ) (Sigma- Aldrich), Adriamycin (0.5 μ m ) (Sigma-Aldrich), and etoposide (10 μ m ) (Sigma-Aldrich).

    Techniques: Chromatin Immunoprecipitation

    Alteration of BiP protein level in response to DNA damage. a HeLa cells were treated with the indicated concentration of thapsigargin (8 h) or etoposide (48 h), and a western blot analysis was performed using the indicated antibody. The

    Journal: Cell Stress & Chaperones

    Article Title: Gene regulatory network of unfolded protein response genes in endoplasmic reticulum stress

    doi: 10.1007/s12192-012-0351-5

    Figure Lengend Snippet: Alteration of BiP protein level in response to DNA damage. a HeLa cells were treated with the indicated concentration of thapsigargin (8 h) or etoposide (48 h), and a western blot analysis was performed using the indicated antibody. The

    Article Snippet: Thapsigargin (Sigma, Saint Louis, MO), tunicamycin (Sigma), etoposide (Wako Pure Chemical Industries, Osaka, Japan), doxorubicin (Wako Pure Chemical Industries), 5-fluorouracil (5-FU, Sigma), proteasome inhibitor I, lactacystin, MG132, ALLN, clasto-lactacystin β-lactone, epoxomicin, ubiquitin aldehyde are purchased from Merck Biosciences (former Calbiochem, Darmstadt, Germany) and were dissolved in dimethyl sulfoxide (DMSO; Wako Pure Chemical Industries) before use.

    Techniques: Concentration Assay, Western Blot

    Transcriptional regulation of UPR genes. a Time-dependent expression changes of BiP mRNA were detected using quantitative RT-PCR in thapsigargin (0.5 μM)-treated HeLa cells. GADH was used as an internal standard. The values represent the

    Journal: Cell Stress & Chaperones

    Article Title: Gene regulatory network of unfolded protein response genes in endoplasmic reticulum stress

    doi: 10.1007/s12192-012-0351-5

    Figure Lengend Snippet: Transcriptional regulation of UPR genes. a Time-dependent expression changes of BiP mRNA were detected using quantitative RT-PCR in thapsigargin (0.5 μM)-treated HeLa cells. GADH was used as an internal standard. The values represent the

    Article Snippet: Thapsigargin (Sigma, Saint Louis, MO), tunicamycin (Sigma), etoposide (Wako Pure Chemical Industries, Osaka, Japan), doxorubicin (Wako Pure Chemical Industries), 5-fluorouracil (5-FU, Sigma), proteasome inhibitor I, lactacystin, MG132, ALLN, clasto-lactacystin β-lactone, epoxomicin, ubiquitin aldehyde are purchased from Merck Biosciences (former Calbiochem, Darmstadt, Germany) and were dissolved in dimethyl sulfoxide (DMSO; Wako Pure Chemical Industries) before use.

    Techniques: Expressing, Quantitative RT-PCR

    Regulation of UPRE by UPR genes. a p5 × ATF6-GL3 was transfected into the cells and 24 h later, thapsigargin (0.5 μM, 8 h) or etoposide (10 μM, 48 h) was added and the luciferase

    Journal: Cell Stress & Chaperones

    Article Title: Gene regulatory network of unfolded protein response genes in endoplasmic reticulum stress

    doi: 10.1007/s12192-012-0351-5

    Figure Lengend Snippet: Regulation of UPRE by UPR genes. a p5 × ATF6-GL3 was transfected into the cells and 24 h later, thapsigargin (0.5 μM, 8 h) or etoposide (10 μM, 48 h) was added and the luciferase

    Article Snippet: Thapsigargin (Sigma, Saint Louis, MO), tunicamycin (Sigma), etoposide (Wako Pure Chemical Industries, Osaka, Japan), doxorubicin (Wako Pure Chemical Industries), 5-fluorouracil (5-FU, Sigma), proteasome inhibitor I, lactacystin, MG132, ALLN, clasto-lactacystin β-lactone, epoxomicin, ubiquitin aldehyde are purchased from Merck Biosciences (former Calbiochem, Darmstadt, Germany) and were dissolved in dimethyl sulfoxide (DMSO; Wako Pure Chemical Industries) before use.

    Techniques: Transfection, Luciferase

    KAP1 phospho-Ser473 after DNA damage is Chk1- and Chk2-dependent . (a) Etoposide-induced KAP1 Ser473 phosphorylation is abolished by Chk1/Chk2 inhibition and reduced upon ATM inhibition. U2OS cells were untreated or treated with 5 μM etoposide (ETP) for 4 h in the presence or absence of KU55933 (ATMi), caffeine (Caff), or AZD7762 (AZD). (b) KAP1 phospho-Ser473 induction after 20 Gy of IR is abolished by AZD7762 (the drug was not removed during the recovery time). Chk2 phospho-Thr68 was used as readout of DNA damage and histone H3 phospho-Ser10 was used as readout for the G2/M checkpoint. (c) AZD7762 decreases KAP1 phospho-Ser473 on immunofluorescence; U2OS cells were treated as in (b). (d) KAP1 Ser473 is targeted by Chk1. U2OS cells were transfected with either siLuc, siChk1, siChk2, or both siChk1 and siChk2, then treated with 10 μM aphidicolin for 1 h. (e) KAP1 Ser473 is targeted by Chk2. U2OS cells were transfected as in (d) and treated as in (b). (f) KAP1 phospho-Ser473 is neither recruited nor excluded from laser-induced DNA-damage sites. Cells were fixed 5, 10 or 30 minutes after micro-irradiation.

    Journal: Genome Biology

    Article Title: A phospho-proteomic screen identifies substrates of the checkpoint kinase Chk1

    doi: 10.1186/gb-2011-12-8-r78

    Figure Lengend Snippet: KAP1 phospho-Ser473 after DNA damage is Chk1- and Chk2-dependent . (a) Etoposide-induced KAP1 Ser473 phosphorylation is abolished by Chk1/Chk2 inhibition and reduced upon ATM inhibition. U2OS cells were untreated or treated with 5 μM etoposide (ETP) for 4 h in the presence or absence of KU55933 (ATMi), caffeine (Caff), or AZD7762 (AZD). (b) KAP1 phospho-Ser473 induction after 20 Gy of IR is abolished by AZD7762 (the drug was not removed during the recovery time). Chk2 phospho-Thr68 was used as readout of DNA damage and histone H3 phospho-Ser10 was used as readout for the G2/M checkpoint. (c) AZD7762 decreases KAP1 phospho-Ser473 on immunofluorescence; U2OS cells were treated as in (b). (d) KAP1 Ser473 is targeted by Chk1. U2OS cells were transfected with either siLuc, siChk1, siChk2, or both siChk1 and siChk2, then treated with 10 μM aphidicolin for 1 h. (e) KAP1 Ser473 is targeted by Chk2. U2OS cells were transfected as in (d) and treated as in (b). (f) KAP1 phospho-Ser473 is neither recruited nor excluded from laser-induced DNA-damage sites. Cells were fixed 5, 10 or 30 minutes after micro-irradiation.

    Article Snippet: Aphidicolin, caffeine, etoposide, hydroxyurea and camptothecin were from Sigma-Aldrich; phleomycin was from Melford Laboratories Ltd., Ipswich, UK.

    Techniques: Inhibition, Immunofluorescence, Transfection, Irradiation

    Effects of HA-BAX on morphology and sub-G 0 content of SW626 cells treated with paclitaxel. Neo-control clone A10 and HA-BAX clone D7 were grown for 48 hr in the presence of medium alone or paclitaxel (0.05 μM), followed by morphologic assessment (Wrights–Giemsa staining) and quantitation of the sub-G 0 fraction by propidium iodide staining as described. Significant amounts of nuclear fragmentation and an increased fraction of sub-G 0 cells are observed for HA-BAX clone D7 compared with neo-control cells, consistent with paclitaxel-mediated apoptosis. Representative data from one of three separate experiments are shown.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: BAX enhances paclitaxel-induced apoptosis through a p53-independent pathway

    doi:

    Figure Lengend Snippet: Effects of HA-BAX on morphology and sub-G 0 content of SW626 cells treated with paclitaxel. Neo-control clone A10 and HA-BAX clone D7 were grown for 48 hr in the presence of medium alone or paclitaxel (0.05 μM), followed by morphologic assessment (Wrights–Giemsa staining) and quantitation of the sub-G 0 fraction by propidium iodide staining as described. Significant amounts of nuclear fragmentation and an increased fraction of sub-G 0 cells are observed for HA-BAX clone D7 compared with neo-control cells, consistent with paclitaxel-mediated apoptosis. Representative data from one of three separate experiments are shown.

    Article Snippet: Propidium iodide used for assessment of DNA content was purchased from Sigma.

    Techniques: Staining, Quantitation Assay

    HMA induces autophagy. ( A ) ARPE 19 cells were untreated or treated with rapamycin for 2 hrs, with HMA or etoposide for 24 hrs and then probed with anti-LC3 antibody. Scale bar 25 μm. ( B ) ARPE-19 cells were treated as before and analysed by Western blot using anti-beclin 1, ATG-7, AGT5-12 and LC3 antibodies. γ tubulin was used as a loading control (left panel). On middle and right panels, cells were treated as before and analysed using anti-ERK and phospho-ERK (*) antibodies (middle panel) or with anti JNK1 or phosphorylated JNK1 (*) antibodies (right panel). Under the western images the quantification of the bands is reported showing a significant increase of Beclin 1 (Means are different from each other as calculated from a one-way anova test ( P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Characterization of stress response in human retinal epithelial cells

    doi: 10.1111/j.1582-4934.2012.01652.x

    Figure Lengend Snippet: HMA induces autophagy. ( A ) ARPE 19 cells were untreated or treated with rapamycin for 2 hrs, with HMA or etoposide for 24 hrs and then probed with anti-LC3 antibody. Scale bar 25 μm. ( B ) ARPE-19 cells were treated as before and analysed by Western blot using anti-beclin 1, ATG-7, AGT5-12 and LC3 antibodies. γ tubulin was used as a loading control (left panel). On middle and right panels, cells were treated as before and analysed using anti-ERK and phospho-ERK (*) antibodies (middle panel) or with anti JNK1 or phosphorylated JNK1 (*) antibodies (right panel). Under the western images the quantification of the bands is reported showing a significant increase of Beclin 1 (Means are different from each other as calculated from a one-way anova test ( P

    Article Snippet: As a positive control of autophagy, cell cultures were treated with 1 μM rapamycin (R0395; Sigma-Aldrich), an inhibitor of mTOR complex, for 2 hrs.

    Techniques: Western Blot

    Role of autophagy in HMA response. ( A ) ARPE-19 cells were treated with HMA in the absence or presence of 1 μM rapamycin or 5 mM 3-MA. After 2 or 4 hrs of treatment, cells were trypsinized and counted. A total of 1000 cells were seeded into 6-well plates. Seven days after seeding, cells were stained with cresyl violet. Controls using rapamycin or 3-MA alone have show no difference with the control (not shown) ( B ) Using image analysis, the surface covered by the cells was measured and plotted. For 2 hrs of treatment all means were significantly different ( P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: Characterization of stress response in human retinal epithelial cells

    doi: 10.1111/j.1582-4934.2012.01652.x

    Figure Lengend Snippet: Role of autophagy in HMA response. ( A ) ARPE-19 cells were treated with HMA in the absence or presence of 1 μM rapamycin or 5 mM 3-MA. After 2 or 4 hrs of treatment, cells were trypsinized and counted. A total of 1000 cells were seeded into 6-well plates. Seven days after seeding, cells were stained with cresyl violet. Controls using rapamycin or 3-MA alone have show no difference with the control (not shown) ( B ) Using image analysis, the surface covered by the cells was measured and plotted. For 2 hrs of treatment all means were significantly different ( P

    Article Snippet: As a positive control of autophagy, cell cultures were treated with 1 μM rapamycin (R0395; Sigma-Aldrich), an inhibitor of mTOR complex, for 2 hrs.

    Techniques: Staining

    Deletion of HtrA2/Omi results in increased sensitivity to mitochondrion-damaging agents. Thymocytes were isolated from control and HtrA2/Omi knockout animals and incubated with increasing concentrations of anti-Fas antibody (A), etoposide (B), or the mitochondrion-damaging agents CCCP (C) and rotenone (D). Viability was determined 16 h after treatment by flow cytometry using propidium iodide. Results are representative of three independent experiments. (E) Viability of simian virus 40 large-T-antigen-immortalized MEFs derived from wild-type (+/+) and HtrA2/Omi knockout animals, determined by measurement of sub-G 1 cell populations by flow cytometry. Cells were incubated in the presence of CCCP (25 μM for 27 h), rotenone (25 μM for 27 h), tunicamycin (2.5 μg/ml for 27 h), or hydrogen peroxide (3 μM for 4.5 h) and compared to untreated control cells. Results show the means ± standard deviations of results of three independent experiments. (F) The neuronalresponse to glutamate-induced cytotoxicity was determined in primary neurons isolated from E14.5 embryos cultured in vitro for 10 days. Following incubation with the indicated concentrations of glutamate for 4 h, cells were stained with Hoechst 33342 and nuclear morphology was determined. Results show the means ± standard deviations of results from two independent experiments where six fields with at least 40 cells were scored for each data point.

    Journal: Molecular and Cellular Biology

    Article Title: Neuroprotective Role of the Reaper-Related Serine Protease HtrA2/Omi Revealed by Targeted Deletion in Mice

    doi: 10.1128/MCB.24.22.9848-9862.2004

    Figure Lengend Snippet: Deletion of HtrA2/Omi results in increased sensitivity to mitochondrion-damaging agents. Thymocytes were isolated from control and HtrA2/Omi knockout animals and incubated with increasing concentrations of anti-Fas antibody (A), etoposide (B), or the mitochondrion-damaging agents CCCP (C) and rotenone (D). Viability was determined 16 h after treatment by flow cytometry using propidium iodide. Results are representative of three independent experiments. (E) Viability of simian virus 40 large-T-antigen-immortalized MEFs derived from wild-type (+/+) and HtrA2/Omi knockout animals, determined by measurement of sub-G 1 cell populations by flow cytometry. Cells were incubated in the presence of CCCP (25 μM for 27 h), rotenone (25 μM for 27 h), tunicamycin (2.5 μg/ml for 27 h), or hydrogen peroxide (3 μM for 4.5 h) and compared to untreated control cells. Results show the means ± standard deviations of results of three independent experiments. (F) The neuronalresponse to glutamate-induced cytotoxicity was determined in primary neurons isolated from E14.5 embryos cultured in vitro for 10 days. Following incubation with the indicated concentrations of glutamate for 4 h, cells were stained with Hoechst 33342 and nuclear morphology was determined. Results show the means ± standard deviations of results from two independent experiments where six fields with at least 40 cells were scored for each data point.

    Article Snippet: Immortalized MEFs were plated (2 × 105 per well) in six-well plates and treated 16 h later with CCCP, rotenone, tunicamycin (Sigma), or hydrogen peroxide (Sigma) for 27 h. DNA content was determined by staining cells with propidium iodide, followed by flow cytometric analysis as previously described ( ).

    Techniques: Isolation, Knock-Out, Incubation, Flow Cytometry, Cytometry, Derivative Assay, Cell Culture, In Vitro, Staining

    Functional analysis of the interaction between ANXA2 and p50 in Mia-Paca2 cells. All data are representative of three independent experiments. ( a ) Interaction between endogenous ANXA2 and p50 in Mia-Paca2 cells. Cell extracts were immunoprecipitated with anti-p50 rabbit polyclonal IgG or anti-rabbit preimmune IgG, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analyzed by western blotting with the indicated antibodies. ( b ) Mammalian expression vectors containing the indicated DNA sequences were introduced into Mia-Paca2 cells by electroporation, and a NF- κ B transcriptional activity assay was performed. ( c ) IL-6 secretion into the culture medium by cells expressing wild-type or Y23A ANXA2 assessed by an enzyme-linked immunosorbent assay. ( d ) Viability (WST reagent) of Mia-Paca2 cells expressing wild-type or Y23A ANXA2 after treatment with TNF- α for 48 h. ( e ) Viability (WST reagent) of Mia-Paca2 cells expressing wild-type or Y23A ANXA2 after treatment with gemcitabine treatment for 48 h. ( f ) Caspase 3 levels and poly ADP-ribose polymerase (PARP) cleavage were confirmed by western blotting analysis after gemcitabine treatment (5 μ M) for 48 h. ( g ) One day before gemcitabine treatment, Mia-Paca2 cells expressing wild-type or Y23A ANXA2 (7 × 10 5 cells) were plated into 100 mm cell culture plate. 1 μ M gemcitabine and 10 μ M Bay 11-7082 were treated for 36 h, and cleaved caspase 3 was confirmed by western blotting analysis. * P

    Journal: Cell Death & Disease

    Article Title: Intracellular annexin A2 regulates NF-κB signaling by binding to the p50 subunit: implications for gemcitabine resistance in pancreatic cancer

    doi: 10.1038/cddis.2014.558

    Figure Lengend Snippet: Functional analysis of the interaction between ANXA2 and p50 in Mia-Paca2 cells. All data are representative of three independent experiments. ( a ) Interaction between endogenous ANXA2 and p50 in Mia-Paca2 cells. Cell extracts were immunoprecipitated with anti-p50 rabbit polyclonal IgG or anti-rabbit preimmune IgG, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and analyzed by western blotting with the indicated antibodies. ( b ) Mammalian expression vectors containing the indicated DNA sequences were introduced into Mia-Paca2 cells by electroporation, and a NF- κ B transcriptional activity assay was performed. ( c ) IL-6 secretion into the culture medium by cells expressing wild-type or Y23A ANXA2 assessed by an enzyme-linked immunosorbent assay. ( d ) Viability (WST reagent) of Mia-Paca2 cells expressing wild-type or Y23A ANXA2 after treatment with TNF- α for 48 h. ( e ) Viability (WST reagent) of Mia-Paca2 cells expressing wild-type or Y23A ANXA2 after treatment with gemcitabine treatment for 48 h. ( f ) Caspase 3 levels and poly ADP-ribose polymerase (PARP) cleavage were confirmed by western blotting analysis after gemcitabine treatment (5 μ M) for 48 h. ( g ) One day before gemcitabine treatment, Mia-Paca2 cells expressing wild-type or Y23A ANXA2 (7 × 10 5 cells) were plated into 100 mm cell culture plate. 1 μ M gemcitabine and 10 μ M Bay 11-7082 were treated for 36 h, and cleaved caspase 3 was confirmed by western blotting analysis. * P

    Article Snippet: The following reagents were used: TNF-α (Sigma, St. Louis, MO, USA), PMA (Sigma), Etoposide (Sigma), Gemcitabine hydrochloride (Sigma), and Bay 11-7082 (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

    Techniques: Functional Assay, Immunoprecipitation, Polyacrylamide Gel Electrophoresis, Western Blot, Expressing, Electroporation, Activity Assay, Enzyme-linked Immunosorbent Assay, Cell Culture

    CREB3L1 activation is independent from DNA breaks. ( A ) On day 0, indicated cells were seeded at 4 × 10 5 cells per 60 mm dish. On day 1, cells were treated with 500 nM doxorubicin or 500 nM etoposide. On day 2, 24 hr after the treatment, the cells were harvested for immunoblot analysis with antibodies reacting against γH2AX or actin. ( B ) Huh7 cells were seeded and treated as described in ( A ). On day 2, cells were separated into nuclear and membrane fractions and analyzed by immunoblot analysis as described in Figure 1B . ( C ) The effect of etoposide on proliferation of the indicated cells was determined as described in Figure 3A . ( D )–( G ) On day 0, indicated cells were seeded at 1.5 × 10 5 cells per 60 mm dish. On day 1, cells were treated with indicated concentrations of etoposide ( D ), doxorubicin ( E ), bleomycin ( F ), or paclitaxel ( G ). On day 3, 48 hr after the treatment, proliferation of the cells was determined by measurement of cellular DNA. The amount of DNA just prior to the drug treatment and after treatment with no drug for 48 hr is set to 0% and 100%, respectively. ( C )–( G ) Results are reported as mean ± S.E.M. of three independent experiments. DOI: http://dx.doi.org/10.7554/eLife.00090.007

    Journal: eLife

    Article Title: Doxorubicin blocks proliferation of cancer cells through proteolytic activation of CREB3L1Decision letterAuthor response

    doi: 10.7554/eLife.00090.011

    Figure Lengend Snippet: CREB3L1 activation is independent from DNA breaks. ( A ) On day 0, indicated cells were seeded at 4 × 10 5 cells per 60 mm dish. On day 1, cells were treated with 500 nM doxorubicin or 500 nM etoposide. On day 2, 24 hr after the treatment, the cells were harvested for immunoblot analysis with antibodies reacting against γH2AX or actin. ( B ) Huh7 cells were seeded and treated as described in ( A ). On day 2, cells were separated into nuclear and membrane fractions and analyzed by immunoblot analysis as described in Figure 1B . ( C ) The effect of etoposide on proliferation of the indicated cells was determined as described in Figure 3A . ( D )–( G ) On day 0, indicated cells were seeded at 1.5 × 10 5 cells per 60 mm dish. On day 1, cells were treated with indicated concentrations of etoposide ( D ), doxorubicin ( E ), bleomycin ( F ), or paclitaxel ( G ). On day 3, 48 hr after the treatment, proliferation of the cells was determined by measurement of cellular DNA. The amount of DNA just prior to the drug treatment and after treatment with no drug for 48 hr is set to 0% and 100%, respectively. ( C )–( G ) Results are reported as mean ± S.E.M. of three independent experiments. DOI: http://dx.doi.org/10.7554/eLife.00090.007

    Article Snippet: Materials We obtained rabbit anti-LSD1 from Cell Signaling (Boston, MA); mouse anti-calnexin from Enzo Life Sciences (Farmingdale, NY); mouse anti-γH2AX from Millipore (Billerica, MA); rabbit anti-Actin and anti-p21 from Abcam (Cambridge, MA); peroxidase-conjugated secondary antibodies from Jackson ImmunoResearch (West Grove, PA); Doxorubicin (Cat# D1515-10MG), bleomycin, etoposide, paclitaxel and N-Hexanoyl-D-sphingosine (C6 -Ceramide) from Sigma-Aldrich (St. Louis, MO); Myriocin and fumonisin B1 from EMD Biosciences (Darmstadt, Germany); and [14 C]palmitate (55 mCi/mmol) from ARC (St. Louis, MO).

    Techniques: Activation Assay

    JMY involved in DNA damage responses in maturing porcine oocyte. A : Typical staining of γ-H2AX in porcine oocytes before and after the treatment with etoposide (25 mg/mL) at MI (A) and MII (B) stage. Red, γ-H2AX; blue, chromatin. B : JMY expression in porcine oocytes on 44 h after etoposide. Note that JMY is localized at nucleus in etoposide treated group (indicated in the arrow). Red, JMY; blue, chromatin. Values represent mean ± s.e.m, * p

    Journal: PLoS ONE

    Article Title: JMY Functions as Actin Nucleation-Promoting Factor and Mediator for p53-Mediated DNA Damage in Porcine Oocytes

    doi: 10.1371/journal.pone.0109385

    Figure Lengend Snippet: JMY involved in DNA damage responses in maturing porcine oocyte. A : Typical staining of γ-H2AX in porcine oocytes before and after the treatment with etoposide (25 mg/mL) at MI (A) and MII (B) stage. Red, γ-H2AX; blue, chromatin. B : JMY expression in porcine oocytes on 44 h after etoposide. Note that JMY is localized at nucleus in etoposide treated group (indicated in the arrow). Red, JMY; blue, chromatin. Values represent mean ± s.e.m, * p

    Article Snippet: To induce DNA damage, 25 mg/mL of etoposide (Sigma) was added into the IVM medium.

    Techniques: Staining, Expressing

    Pregnenolone sulphate-mediated insulin secretion is inhibited by mefenamic acid. INS-1E cells were cultured and insulin secretion measured as described in Methods . Test substances were added as indicated. (A) The effect of pregnenolone sulphate and mefenamic

    Journal: British Journal of Pharmacology

    Article Title: Fenamates as TRP channel blockers: mefenamic acid selectively blocks TRPM3

    doi: 10.1111/j.1476-5381.2010.01186.x

    Figure Lengend Snippet: Pregnenolone sulphate-mediated insulin secretion is inhibited by mefenamic acid. INS-1E cells were cultured and insulin secretion measured as described in Methods . Test substances were added as indicated. (A) The effect of pregnenolone sulphate and mefenamic

    Article Snippet: Flufenamic acid, niflumic acid, mefenamic acid, meclofenamic acid, S645648, tolfenamic acid and pregnenolone sulphate (Sigma-Aldrich, Deisenhofen, Germany) were diluted from 50 mM stock solutions in DMSO.

    Techniques: Cell Culture

    Effects of mefenamic acid on primary mouse pancreatic β-cells. Changes in [Ca 2+ ] i are depicted by the fluorescence ratios F 340 / F 380 of Fura-2-loaded primary mouse pancreatic islet cells. (A) Measurement of [Ca 2+ ] i after stimulation with 35 µM

    Journal: British Journal of Pharmacology

    Article Title: Fenamates as TRP channel blockers: mefenamic acid selectively blocks TRPM3

    doi: 10.1111/j.1476-5381.2010.01186.x

    Figure Lengend Snippet: Effects of mefenamic acid on primary mouse pancreatic β-cells. Changes in [Ca 2+ ] i are depicted by the fluorescence ratios F 340 / F 380 of Fura-2-loaded primary mouse pancreatic islet cells. (A) Measurement of [Ca 2+ ] i after stimulation with 35 µM

    Article Snippet: Flufenamic acid, niflumic acid, mefenamic acid, meclofenamic acid, S645648, tolfenamic acid and pregnenolone sulphate (Sigma-Aldrich, Deisenhofen, Germany) were diluted from 50 mM stock solutions in DMSO.

    Techniques: Fluorescence

    Effects of mefenamic acid on pancreatic β-cells. A. Western blot analysis of membranes extracted from INS-1E cells, HEK293 control cells (con) and TRPM3-expressing HEK293 cells (TRPM3). The anti-TRPM3 antibody detected two bands of approx. 150

    Journal: British Journal of Pharmacology

    Article Title: Fenamates as TRP channel blockers: mefenamic acid selectively blocks TRPM3

    doi: 10.1111/j.1476-5381.2010.01186.x

    Figure Lengend Snippet: Effects of mefenamic acid on pancreatic β-cells. A. Western blot analysis of membranes extracted from INS-1E cells, HEK293 control cells (con) and TRPM3-expressing HEK293 cells (TRPM3). The anti-TRPM3 antibody detected two bands of approx. 150

    Article Snippet: Flufenamic acid, niflumic acid, mefenamic acid, meclofenamic acid, S645648, tolfenamic acid and pregnenolone sulphate (Sigma-Aldrich, Deisenhofen, Germany) were diluted from 50 mM stock solutions in DMSO.

    Techniques: Western Blot, Expressing

    pH-dependence of mefenamic acid on the inhibition of TRPM3-mediated currents. (A) Time course of TRPM3 current at membrane potentials of −80 (lower trace) and +80 mV (upper trace); the pH value of the extracellular solution was 8 before the mefenamic

    Journal: British Journal of Pharmacology

    Article Title: Fenamates as TRP channel blockers: mefenamic acid selectively blocks TRPM3

    doi: 10.1111/j.1476-5381.2010.01186.x

    Figure Lengend Snippet: pH-dependence of mefenamic acid on the inhibition of TRPM3-mediated currents. (A) Time course of TRPM3 current at membrane potentials of −80 (lower trace) and +80 mV (upper trace); the pH value of the extracellular solution was 8 before the mefenamic

    Article Snippet: Flufenamic acid, niflumic acid, mefenamic acid, meclofenamic acid, S645648, tolfenamic acid and pregnenolone sulphate (Sigma-Aldrich, Deisenhofen, Germany) were diluted from 50 mM stock solutions in DMSO.

    Techniques: Inhibition

    Extracellular application of mefenamic acid inhibits TRPM3 currents in transfected HEK293 cells. (A) Current of TRPM3 at membrane potentials of −80 (lower trace) and +80 mV (upper trace) during the application of the TRPM3 activator pregnenolone

    Journal: British Journal of Pharmacology

    Article Title: Fenamates as TRP channel blockers: mefenamic acid selectively blocks TRPM3

    doi: 10.1111/j.1476-5381.2010.01186.x

    Figure Lengend Snippet: Extracellular application of mefenamic acid inhibits TRPM3 currents in transfected HEK293 cells. (A) Current of TRPM3 at membrane potentials of −80 (lower trace) and +80 mV (upper trace) during the application of the TRPM3 activator pregnenolone

    Article Snippet: Flufenamic acid, niflumic acid, mefenamic acid, meclofenamic acid, S645648, tolfenamic acid and pregnenolone sulphate (Sigma-Aldrich, Deisenhofen, Germany) were diluted from 50 mM stock solutions in DMSO.

    Techniques: Transfection

    ANK1 is upregulated following exposure to different inducers of DNA damage and in a variety of cell types. ( a ) Diagram showing the location of miR-486 in relation to the open reading frame of the cytoskeleton adaptor gene ANK1 , as well as the small ankyrin-1 isoform (sAnk1). MiR-486 is located in the intronic region between the last two exons of the ANK1 gene. ( b ) MCF10A cells were treated with doxorubicin (dox.) as in Figure 1c , and ANK1 mRNA levels were quantified by qPCR. The p53-regulated p21 (Waf1/ Cip1) transcript served as a positive control ( n =3). ( c ) Representative western analysis of protein samples harvested from cells undergoing DNA damage ( n =3). The ankyrin-1 protein (ANK1) (246 kDa) was quantified (right panel) and normalised to β -actin levels. ( d ) Ankyrin-1 was induced following ionising radiation (IR, 10 Gy) exposure in MCF10A cells but not by ultraviolet light-C (UVC, 100 J/m 2 ). Cells were harvested 24 h after IR/UVC exposure. Cisplatin (10 μ g/ml) and mitomycin C (300 nM) treatment for 24 h also induced ankyrin-1. Representative western blots are shown ( n =3). ( e ) Ankyrin-1 was upregulated following 25 μ M etoposide (etop.) treatment for 24 h in retinal pigment epithelium (RPE) and MCF7 cells, and in A549 cells following IR (5 Gy) exposure where cells were harvested after 6 h. Representative western blots are shown ( n =3). For all bar charts, values are mean±S.D. ( t -test, n =3, compared with 0 h control), * P

    Journal: Cell Death & Disease

    Article Title: The cytoskeleton adaptor protein ankyrin-1 is upregulated by p53 following DNA damage and alters cell migration

    doi: 10.1038/cddis.2016.91

    Figure Lengend Snippet: ANK1 is upregulated following exposure to different inducers of DNA damage and in a variety of cell types. ( a ) Diagram showing the location of miR-486 in relation to the open reading frame of the cytoskeleton adaptor gene ANK1 , as well as the small ankyrin-1 isoform (sAnk1). MiR-486 is located in the intronic region between the last two exons of the ANK1 gene. ( b ) MCF10A cells were treated with doxorubicin (dox.) as in Figure 1c , and ANK1 mRNA levels were quantified by qPCR. The p53-regulated p21 (Waf1/ Cip1) transcript served as a positive control ( n =3). ( c ) Representative western analysis of protein samples harvested from cells undergoing DNA damage ( n =3). The ankyrin-1 protein (ANK1) (246 kDa) was quantified (right panel) and normalised to β -actin levels. ( d ) Ankyrin-1 was induced following ionising radiation (IR, 10 Gy) exposure in MCF10A cells but not by ultraviolet light-C (UVC, 100 J/m 2 ). Cells were harvested 24 h after IR/UVC exposure. Cisplatin (10 μ g/ml) and mitomycin C (300 nM) treatment for 24 h also induced ankyrin-1. Representative western blots are shown ( n =3). ( e ) Ankyrin-1 was upregulated following 25 μ M etoposide (etop.) treatment for 24 h in retinal pigment epithelium (RPE) and MCF7 cells, and in A549 cells following IR (5 Gy) exposure where cells were harvested after 6 h. Representative western blots are shown ( n =3). For all bar charts, values are mean±S.D. ( t -test, n =3, compared with 0 h control), * P

    Article Snippet: DNA damage and p53 induction DNA damage was achieved using either 25 μ M etoposide (24 h) (Cayman, Ann Arbor, MI, USA) in dimethyl suphoxide (DMSO), 400 nM doxorubicin hydrochloride (0–48 h) (Sigma, St. Louis, MO, USA), 10 μ g/ml cisplatin (24 h) (Sigma) or 300 nM mitomycin C (24 h) (Sigma).

    Techniques: Real-time Polymerase Chain Reaction, Positive Control, Western Blot