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Data supporting the inability of indomethacin to induce autophagy in U251 glioma cells
Data in Brief ∎ (∎∎∎∎) ∎∎∎–∎∎∎
1 Contents lists available at ScienceDirect 2 3 4 5 6 journal homepage: www.elsevier.com/locate/dib 7 8 9 Data Article 10 11 12 13 14 15 Aleksandar Pantovic a, Katarina Arsikin b, Milica Kosic b, 16 Biljana Ristic b, Vladimir Trajkovic b,n, 17 c,n 18 Q1 Ljubica Harhaji-Trajkovic 19 a Neurology Clinic, Military Medical Academy, Belgrade, Serbia b 20 Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade, Serbia 21 c Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Despot Stefan Blvd. 142, 22 11000 Belgrade, Serbia 23 24 25 a r t i c l e i n f o abstract 26 27 Article history: Autophagy, a catabolic process involving intracellular degradation of 28 Received 16 December 2016 unnecessary or dysfunctional cellular components through the 29 Received in revised form lysosomal machinery, could act as a prosurvival, as well as a cyto30 20 January 2017 toxic mechanism (K.R. Parzych, D.J. Klionsky, 2014) [1]. Cycloox- Q2 Accepted 6 February 2017 31 ygenase inhibitor indomethacin inhibits proliferation of glioma 32 cells, and has been reported to reduce the activity of the main Keywords: 33 autophagy repressor mammalian target of rapamycin (mTOR) Indomethacin 34 (A. Pantovic, M. Bosnjak, K. Arsikin, M. Kosic, M. Mandic, Glioma B. Ristic, J. Tosic, D. Grujicic, A. Isakovic, N. Micic, V. Trajkovic, 35 Autophagy L. Harhaji-Trajkovic, 2016) [2]. Here we investigated the ability of 36 LC3 indomethacin to induce autophagy in U251 human glioma cells. We 37 Beclin-1 assessed the influence of indomethacin on intracellular acidifica38 tion, expression of proautophagic protein beclin-1, and conversion 39 of microtubule-associated protein light chain 3-I (LC3-I) to 40 autophagosome-associated LC3-II, in the presence or absence of 41 lysosomal inhibitors. The effect of genetic and pharmacologi42 cal downregulation of autophagy on the cytotoxicity of indo43 methacin was evaluated. The interpretation of these data can be 44 found in “in vitro antiglioma action of indomethacin is mediated 45 46 47 48 49 DOI of original article: http://dx.doi.org/10.1016/j.biocel.2016.12.007 n Corresponding authors. 50 E-mail addresses: [email protected] (V. Trajkovic), [email protected] (L. Harhaji-Trajkovic). 51 52 http://dx.doi.org/10.1016/j.dib.2017.02.012 53 2352-3409/& 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license 54 (http://creativecommons.org/licenses/by/4.0/).
Data in Brief
Data supporting the inability of indomethacin to induce autophagy in U251 glioma cells
Please cite this article as: A. Pantovic, et al., Data supporting the inability of indomethacin to induce autophagy in U251 glioma cells, Data in Brief (2017), http://dx.doi.org/10.1016/j. dib.2017.02.012i
A. Pantovic et al. / Data in Brief ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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via AMP-activated protein kinase/mTOR complex 1 signaling pathway” (Pantovic et al., 2016; http://dx.doi.org/10.1016/j.biocel.2016. 12.007) [2]. & 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Specifications Table Subject area More specific subject area Type of data How data was acquired Data format Experimental factors Experimental features Data source location Data accessibility
Biology Glioma, autophagy, indomethacin Figure Flow cytometry, immunoblot, siRNA transfection, and MTT viability test Analyzed U251 glioma cells were treated with indomethacin and bafilomycin A1, chloroquine, wortmannin, or ammonium chloride, or transfected with LC3, beclin-1, or control siRNA before treatment with indomethacin After indomethacin treatment viability of U251 cells was determined, or the cells were prepared for the flow cytometric and immunoblot assessment of autophagy markers School of Medicine, Belgrade, Serbia All data are provided with this article
Value of the data
The data provide the assessment of autophagy-inducing ability of indomethacin in glioma cells. The data provide the assessment of the effect of pharmacological and genetic inhibition of autophagy on the cytotoxic action of indomethacin.
These data might be valuable for researchers interested in autophagy-modulating effects of cyclooxygenase inhibitors.
These data might be valuable for researchers interested in anticancer and antiglioma effects of indomethacin.
1. Data To investigate the ability of indomethacin to induce autophagy in U251 glioma cells, we assessed its influence on intracellular acidification, expression of beclin-1, and conversion of microtubuleassociated protein light chain 3 (LC3) as autophagy hallmarks. Flow cytometric analysis of pHsensitive acridine orange fluorescence revealed that indomethacin failed to increase intracellular acidification (Fig. 1A). Furthermore, the drug did not increase the expression of proautophagic protein beclin-1 or conversion of LC3-I to its lipidated, autophagosome-associated LC3-II form (Fig. 2B). Since the inability of indomethacin to augment LC3-II levels could result from the increase in LC3 degradation in autophagolysosomes [3], we analyzed LC3-II concentration in cells in which autophagic proteolysis was blocked by lysosomal inhibitors chloroquine, bafilomycin A1, or ammonium chloride. While proteolysis inhibitors expectedly increased the levels of LC3-II when applied alone, no additional increase was detected in the presence of indomethacin (Fig. 1C), thus confirming its inability to Please cite this article as: A. Pantovic, et al., Data supporting the inability of indomethacin to induce autophagy in U251 glioma cells, Data in Brief (2017), http://dx.doi.org/10.1016/j. dib.2017.02.012i
A. Pantovic et al. / Data in Brief ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Fig. 1. The effect of indomethacin of the expression of autophagy markers in U251 glioma cells. (A) U251 cells were incubated with indomethacin (250 mM) for 24 h. The intracellular acidification was determined by flow cytometry as a ratio of acridine orange (AO) red/green (FL3/FL1) mean fluorescence intensity, and the representative flow cytometry histograms of three independent experiments are shown. (B) U251 cells were incubated with indomethacin (250 mM) for the indicated time periods, and beclin-1 and LC3 levels were analyzed by immunoblotting. (C) Autophagic flux was assessed by examining LC3 conversion in U251 cells incubated with indomethacin (250 mM) for 8 h, in the presence or absence or proteolysis inhibitors chloroquine (CQ; 20 mM), ammonium chloride (AC; 20 mM), or bafilomycin A1 (BAF; 20 nm). (B, C) The representative blots are shown, and the data are presented as mean 7SD values of three independent experiments (*p o 0.05 compared to untreated cells).
increase autophagic flux. We next employed genetic and pharmacological approaches to assess the role of autophagy in indomethacin-mediated cytotoxicity to U251 cells. RNA interference-mediated downregulation of autophagy-essential genes LC3 and beclin-1 (the knockdown efficiency confirmed by immunoblot in Fig. 2A) had no effect on the viability of indomethacin-treated U251 cells (Fig. 2B). Accordingly, the suppression of autophagy by proton-pump blocker bafilomycin A1, lysosomal inhibitor chloroquine, or class III phosphoinositide 3-kinase inhibitor wortmannin [3] failed to affect antiglioma effect of indomethacin (Fig. 2C).
2. Experimental design, materials and methods 2.1. Cells and cell culture The human glioma cell line U251-MG was obtained by the European Collection of Authenticated Cell Cultures (ECACC 09063001), and maintained in 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)-buffered Roswell Park Memorial Institute (RPMI) 1640 cell culture medium supplemented with 2 mM L-glutamine, antibiotic/antimycotic mixture (1%), and 10% fetal bovine Please cite this article as: A. Pantovic, et al., Data supporting the inability of indomethacin to induce autophagy in U251 glioma cells, Data in Brief (2017), http://dx.doi.org/10.1016/j. dib.2017.02.012i
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A. Pantovic et al. / Data in Brief ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Fig. 2. The effect of genetic and pharmacological inhibition of autophagy on indomethacin cytotoxicity. (A, B) U251 cells were transfected with control, beclin (BCN1), or LC3 siRNA. The knockdown efficiency is assessed by immunoblot (A). The siRNAtransfected cells were incubated with different concentrations of indomethacin for 24 h, and the cell viability was assessed by MTT assay (B). (C) U251 cells were treated for 24 h with indomethacin (250 mM) in the presence or absence of autophagy inhibitors bafilomycin A1 (BAF; 20 nM), chloroquine (CQ; 20 mM), or wortmannin (WORT; 200 nM), and cell viability was analyzed by MTT assay. (A-C) The data are presented as mean7 SD values of at least three independent experiments (*p o 0.05).
serum (all from PAA, Pasching, Austria). U251 glioma cells before passage 10 were incubated in 96well flat bottom plates (1 104 cells/well) for the cell viability assays, 24-well plates (1 105 cells/ well) for the flow cytometry analysis, or 90 mm Petri dishes (2 106 cells) for the immunoblotting. Cells were rested for 24 h and then treated with indomethacin (Sigma-Aldrich, St. Louis, MO) in the presence or absence of bafilomycin A1, chloroquine, ammonium chloride, or wortmannin (all from
Please cite this article as: A. Pantovic, et al., Data supporting the inability of indomethacin to induce autophagy in U251 glioma cells, Data in Brief (2017), http://dx.doi.org/10.1016/j. dib.2017.02.012i
A. Pantovic et al. / Data in Brief ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Sigma-Aldrich, St. Louis, MO). The incubation times and concentrations of agents are stated in figure captions and/or figures. 2.2. Determination of cell viability and cell death The cell viability was determined exactly as previously described, using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay to measure the activity of mitochondrial dehydrogenases [4]. The results were presented as % of the cell viability in untreated cells. 2.3. Intracellular acidification measurement The changes in intracellular acidification were assessed by supravital staining with acridine orange (Sigma-Aldrich, St. Louis, MO), a pH-sensitive dye that shifts its fluorescence from green to red with decreasing pH. Acridine orange-stained cells (1 mM, 15 min, 37 °C) were analyzed using FACSCalibur flow cytometer (BD Biosciences, Heidelberg, Germany) and CellQuest Pro software, and intracellular acidification was quantified as a red/green fluorescence ratio (mean FL3/FL1) arbitrarily set to 1 in control samples. 2.4. Immunoblotting Cells were lysed by 30 min incubation on ice in lysis buffer (30 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% NP-40) containing protease/phosphatase inhibitor cocktail (all from Sigma-Aldrich, St. Louis, MO). The cell lysates were centrifuged at 14000 g for 15 min at 4 °C, and the supernatants were collected. Equal protein amounts from each sample were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad, Marnes-la-Coquette, France). Following incubation with antibodies against LC3B, beclin-1, and actin (all from Cell Signaling Technology, Beverly, MA) as primary antibodies, and peroxidase-conjugated goat anti-rabbit IgG (Jackson IP Laboratories, West Grove, PA) as a secondary antibody, specific protein bands were visualized using enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, Piscataway, NJ). The protein levels were quantified by densitometry using ImageJ software (NIH, Bethesda, MD), and expressed relative to actin as a loading control. The results are presented as fold change in signal intensity, which was arbitrarily set to 1 in untreated cells. 2.5. RNA interference The transfection of subconfluent U251 cells with small interfering RNA (siRNA) targeting human beclin-1, LC3B, or scrambled control siRNA (FlexiTube GeneSolution with 4 preselected siRNAs for each gene; Qiagen, Valencia, CA) was performed using Lipofectamine (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. After transfection, cells were grown for 24 h before treatment with indomethacin. 2.6. Statistical analysis The statistical significance of the differences was analyzed by one-way analysis of variance (ANOVA) followed by Student-Newman–Keuls test. A p value of less than 0.05 was considered significant. Each experiment was performed in triplicates and the data are presented as mean 7SD from at least three independent experiments.
Acknowledgments This work was supported by the Ministry of Education, Science, and Technological Development of the Republic of Serbia (grants 173053 and 41025). Please cite this article as: A. Pantovic, et al., Data supporting the inability of indomethacin to induce autophagy in U251 glioma cells, Data in Brief (2017), http://dx.doi.org/10.1016/j. dib.2017.02.012i
A. Pantovic et al. / Data in Brief ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Transparency document. Supplementary material Transparency data associated with this article can be found in the online version at http://dx.doi. org/10.1016/j.dib.2017.02.012.
References [1] K.R. Parzych, D.J. Klionsky, An overview of autophagy: morphology, mechanism, and regulation, Antioxid. Redox Signal 20 (2014) 460–473. [2] A. Pantovic, M. Bosnjak, K. Arsikin, M. Kosic, M. Mandic, B. Ristic, J. Tosic, D. Grujicic, A. Isakovic, N. Micic, V. Trajkovic, L. Harhaji-Trajkovic, in vitro antiglioma action of indomethacin is mediated via AMP-activated protein kinase/mTOR complex 1 signalling pathway, Int. J. Biochem. Cell Biol. 83 (2016) 84–96. [3] N. Mizushima, T. Yoshimori, B. Levine, Methods in mammalian autophagy research, Cell 140 (2010) 313–326. [4] G.N. Kaludjerovic, D. Miljkovic, M. Momcilovic, V.M. Djinovic, M. Mostarica Stojkovic, T.J. Sabo, V. Trajkovic, Novel platinum (IV) complexes induce rapid tumor cell death in vitro, Int. J. Cancer 116 (2005) 479–486.
Please cite this article as: A. Pantovic, et al., Data supporting the inability of indomethacin to induce autophagy in U251 glioma cells, Data in Brief (2017), http://dx.doi.org/10.1016/j. dib.2017.02.012i
1 Contents lists available at ScienceDirect 2 3 4 5 6 journal homepage: www.elsevier.com/locate/dib 7 8 9 Data Article 10 11 12 13 14 15 Aleksandar Pantovic a, Katarina Arsikin b, Milica Kosic b, 16 Biljana Ristic b, Vladimir Trajkovic b,n, 17 c,n 18 Q1 Ljubica Harhaji-Trajkovic 19 a Neurology Clinic, Military Medical Academy, Belgrade, Serbia b 20 Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000 Belgrade, Serbia 21 c Institute for Biological Research "Sinisa Stankovic", University of Belgrade, Despot Stefan Blvd. 142, 22 11000 Belgrade, Serbia 23 24 25 a r t i c l e i n f o abstract 26 27 Article history: Autophagy, a catabolic process involving intracellular degradation of 28 Received 16 December 2016 unnecessary or dysfunctional cellular components through the 29 Received in revised form lysosomal machinery, could act as a prosurvival, as well as a cyto30 20 January 2017 toxic mechanism (K.R. Parzych, D.J. Klionsky, 2014) [1]. Cycloox- Q2 Accepted 6 February 2017 31 ygenase inhibitor indomethacin inhibits proliferation of glioma 32 cells, and has been reported to reduce the activity of the main Keywords: 33 autophagy repressor mammalian target of rapamycin (mTOR) Indomethacin 34 (A. Pantovic, M. Bosnjak, K. Arsikin, M. Kosic, M. Mandic, Glioma B. Ristic, J. Tosic, D. Grujicic, A. Isakovic, N. Micic, V. Trajkovic, 35 Autophagy L. Harhaji-Trajkovic, 2016) [2]. Here we investigated the ability of 36 LC3 indomethacin to induce autophagy in U251 human glioma cells. We 37 Beclin-1 assessed the influence of indomethacin on intracellular acidifica38 tion, expression of proautophagic protein beclin-1, and conversion 39 of microtubule-associated protein light chain 3-I (LC3-I) to 40 autophagosome-associated LC3-II, in the presence or absence of 41 lysosomal inhibitors. The effect of genetic and pharmacologi42 cal downregulation of autophagy on the cytotoxicity of indo43 methacin was evaluated. The interpretation of these data can be 44 found in “in vitro antiglioma action of indomethacin is mediated 45 46 47 48 49 DOI of original article: http://dx.doi.org/10.1016/j.biocel.2016.12.007 n Corresponding authors. 50 E-mail addresses: [email protected] (V. Trajkovic), [email protected] (L. Harhaji-Trajkovic). 51 52 http://dx.doi.org/10.1016/j.dib.2017.02.012 53 2352-3409/& 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license 54 (http://creativecommons.org/licenses/by/4.0/).
Data in Brief
Data supporting the inability of indomethacin to induce autophagy in U251 glioma cells
Please cite this article as: A. Pantovic, et al., Data supporting the inability of indomethacin to induce autophagy in U251 glioma cells, Data in Brief (2017), http://dx.doi.org/10.1016/j. dib.2017.02.012i
A. Pantovic et al. / Data in Brief ∎ (∎∎∎∎) ∎∎∎–∎∎∎
2
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via AMP-activated protein kinase/mTOR complex 1 signaling pathway” (Pantovic et al., 2016; http://dx.doi.org/10.1016/j.biocel.2016. 12.007) [2]. & 2017 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Specifications Table Subject area More specific subject area Type of data How data was acquired Data format Experimental factors Experimental features Data source location Data accessibility
Biology Glioma, autophagy, indomethacin Figure Flow cytometry, immunoblot, siRNA transfection, and MTT viability test Analyzed U251 glioma cells were treated with indomethacin and bafilomycin A1, chloroquine, wortmannin, or ammonium chloride, or transfected with LC3, beclin-1, or control siRNA before treatment with indomethacin After indomethacin treatment viability of U251 cells was determined, or the cells were prepared for the flow cytometric and immunoblot assessment of autophagy markers School of Medicine, Belgrade, Serbia All data are provided with this article
Value of the data
The data provide the assessment of autophagy-inducing ability of indomethacin in glioma cells. The data provide the assessment of the effect of pharmacological and genetic inhibition of autophagy on the cytotoxic action of indomethacin.
These data might be valuable for researchers interested in autophagy-modulating effects of cyclooxygenase inhibitors.
These data might be valuable for researchers interested in anticancer and antiglioma effects of indomethacin.
1. Data To investigate the ability of indomethacin to induce autophagy in U251 glioma cells, we assessed its influence on intracellular acidification, expression of beclin-1, and conversion of microtubuleassociated protein light chain 3 (LC3) as autophagy hallmarks. Flow cytometric analysis of pHsensitive acridine orange fluorescence revealed that indomethacin failed to increase intracellular acidification (Fig. 1A). Furthermore, the drug did not increase the expression of proautophagic protein beclin-1 or conversion of LC3-I to its lipidated, autophagosome-associated LC3-II form (Fig. 2B). Since the inability of indomethacin to augment LC3-II levels could result from the increase in LC3 degradation in autophagolysosomes [3], we analyzed LC3-II concentration in cells in which autophagic proteolysis was blocked by lysosomal inhibitors chloroquine, bafilomycin A1, or ammonium chloride. While proteolysis inhibitors expectedly increased the levels of LC3-II when applied alone, no additional increase was detected in the presence of indomethacin (Fig. 1C), thus confirming its inability to Please cite this article as: A. Pantovic, et al., Data supporting the inability of indomethacin to induce autophagy in U251 glioma cells, Data in Brief (2017), http://dx.doi.org/10.1016/j. dib.2017.02.012i
A. Pantovic et al. / Data in Brief ∎ (∎∎∎∎) ∎∎∎–∎∎∎
109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162
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Fig. 1. The effect of indomethacin of the expression of autophagy markers in U251 glioma cells. (A) U251 cells were incubated with indomethacin (250 mM) for 24 h. The intracellular acidification was determined by flow cytometry as a ratio of acridine orange (AO) red/green (FL3/FL1) mean fluorescence intensity, and the representative flow cytometry histograms of three independent experiments are shown. (B) U251 cells were incubated with indomethacin (250 mM) for the indicated time periods, and beclin-1 and LC3 levels were analyzed by immunoblotting. (C) Autophagic flux was assessed by examining LC3 conversion in U251 cells incubated with indomethacin (250 mM) for 8 h, in the presence or absence or proteolysis inhibitors chloroquine (CQ; 20 mM), ammonium chloride (AC; 20 mM), or bafilomycin A1 (BAF; 20 nm). (B, C) The representative blots are shown, and the data are presented as mean 7SD values of three independent experiments (*p o 0.05 compared to untreated cells).
increase autophagic flux. We next employed genetic and pharmacological approaches to assess the role of autophagy in indomethacin-mediated cytotoxicity to U251 cells. RNA interference-mediated downregulation of autophagy-essential genes LC3 and beclin-1 (the knockdown efficiency confirmed by immunoblot in Fig. 2A) had no effect on the viability of indomethacin-treated U251 cells (Fig. 2B). Accordingly, the suppression of autophagy by proton-pump blocker bafilomycin A1, lysosomal inhibitor chloroquine, or class III phosphoinositide 3-kinase inhibitor wortmannin [3] failed to affect antiglioma effect of indomethacin (Fig. 2C).
2. Experimental design, materials and methods 2.1. Cells and cell culture The human glioma cell line U251-MG was obtained by the European Collection of Authenticated Cell Cultures (ECACC 09063001), and maintained in 20 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)-buffered Roswell Park Memorial Institute (RPMI) 1640 cell culture medium supplemented with 2 mM L-glutamine, antibiotic/antimycotic mixture (1%), and 10% fetal bovine Please cite this article as: A. Pantovic, et al., Data supporting the inability of indomethacin to induce autophagy in U251 glioma cells, Data in Brief (2017), http://dx.doi.org/10.1016/j. dib.2017.02.012i
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A. Pantovic et al. / Data in Brief ∎ (∎∎∎∎) ∎∎∎–∎∎∎
Fig. 2. The effect of genetic and pharmacological inhibition of autophagy on indomethacin cytotoxicity. (A, B) U251 cells were transfected with control, beclin (BCN1), or LC3 siRNA. The knockdown efficiency is assessed by immunoblot (A). The siRNAtransfected cells were incubated with different concentrations of indomethacin for 24 h, and the cell viability was assessed by MTT assay (B). (C) U251 cells were treated for 24 h with indomethacin (250 mM) in the presence or absence of autophagy inhibitors bafilomycin A1 (BAF; 20 nM), chloroquine (CQ; 20 mM), or wortmannin (WORT; 200 nM), and cell viability was analyzed by MTT assay. (A-C) The data are presented as mean7 SD values of at least three independent experiments (*p o 0.05).
serum (all from PAA, Pasching, Austria). U251 glioma cells before passage 10 were incubated in 96well flat bottom plates (1 104 cells/well) for the cell viability assays, 24-well plates (1 105 cells/ well) for the flow cytometry analysis, or 90 mm Petri dishes (2 106 cells) for the immunoblotting. Cells were rested for 24 h and then treated with indomethacin (Sigma-Aldrich, St. Louis, MO) in the presence or absence of bafilomycin A1, chloroquine, ammonium chloride, or wortmannin (all from
Please cite this article as: A. Pantovic, et al., Data supporting the inability of indomethacin to induce autophagy in U251 glioma cells, Data in Brief (2017), http://dx.doi.org/10.1016/j. dib.2017.02.012i
A. Pantovic et al. / Data in Brief ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Sigma-Aldrich, St. Louis, MO). The incubation times and concentrations of agents are stated in figure captions and/or figures. 2.2. Determination of cell viability and cell death The cell viability was determined exactly as previously described, using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay to measure the activity of mitochondrial dehydrogenases [4]. The results were presented as % of the cell viability in untreated cells. 2.3. Intracellular acidification measurement The changes in intracellular acidification were assessed by supravital staining with acridine orange (Sigma-Aldrich, St. Louis, MO), a pH-sensitive dye that shifts its fluorescence from green to red with decreasing pH. Acridine orange-stained cells (1 mM, 15 min, 37 °C) were analyzed using FACSCalibur flow cytometer (BD Biosciences, Heidelberg, Germany) and CellQuest Pro software, and intracellular acidification was quantified as a red/green fluorescence ratio (mean FL3/FL1) arbitrarily set to 1 in control samples. 2.4. Immunoblotting Cells were lysed by 30 min incubation on ice in lysis buffer (30 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% NP-40) containing protease/phosphatase inhibitor cocktail (all from Sigma-Aldrich, St. Louis, MO). The cell lysates were centrifuged at 14000 g for 15 min at 4 °C, and the supernatants were collected. Equal protein amounts from each sample were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad, Marnes-la-Coquette, France). Following incubation with antibodies against LC3B, beclin-1, and actin (all from Cell Signaling Technology, Beverly, MA) as primary antibodies, and peroxidase-conjugated goat anti-rabbit IgG (Jackson IP Laboratories, West Grove, PA) as a secondary antibody, specific protein bands were visualized using enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, Piscataway, NJ). The protein levels were quantified by densitometry using ImageJ software (NIH, Bethesda, MD), and expressed relative to actin as a loading control. The results are presented as fold change in signal intensity, which was arbitrarily set to 1 in untreated cells. 2.5. RNA interference The transfection of subconfluent U251 cells with small interfering RNA (siRNA) targeting human beclin-1, LC3B, or scrambled control siRNA (FlexiTube GeneSolution with 4 preselected siRNAs for each gene; Qiagen, Valencia, CA) was performed using Lipofectamine (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. After transfection, cells were grown for 24 h before treatment with indomethacin. 2.6. Statistical analysis The statistical significance of the differences was analyzed by one-way analysis of variance (ANOVA) followed by Student-Newman–Keuls test. A p value of less than 0.05 was considered significant. Each experiment was performed in triplicates and the data are presented as mean 7SD from at least three independent experiments.
Acknowledgments This work was supported by the Ministry of Education, Science, and Technological Development of the Republic of Serbia (grants 173053 and 41025). Please cite this article as: A. Pantovic, et al., Data supporting the inability of indomethacin to induce autophagy in U251 glioma cells, Data in Brief (2017), http://dx.doi.org/10.1016/j. dib.2017.02.012i
A. Pantovic et al. / Data in Brief ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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271 272 273 274 275 276 277 278 279 280 281 282 283 284
Transparency document. Supplementary material Transparency data associated with this article can be found in the online version at http://dx.doi. org/10.1016/j.dib.2017.02.012.
References [1] K.R. Parzych, D.J. Klionsky, An overview of autophagy: morphology, mechanism, and regulation, Antioxid. Redox Signal 20 (2014) 460–473. [2] A. Pantovic, M. Bosnjak, K. Arsikin, M. Kosic, M. Mandic, B. Ristic, J. Tosic, D. Grujicic, A. Isakovic, N. Micic, V. Trajkovic, L. Harhaji-Trajkovic, in vitro antiglioma action of indomethacin is mediated via AMP-activated protein kinase/mTOR complex 1 signalling pathway, Int. J. Biochem. Cell Biol. 83 (2016) 84–96. [3] N. Mizushima, T. Yoshimori, B. Levine, Methods in mammalian autophagy research, Cell 140 (2010) 313–326. [4] G.N. Kaludjerovic, D. Miljkovic, M. Momcilovic, V.M. Djinovic, M. Mostarica Stojkovic, T.J. Sabo, V. Trajkovic, Novel platinum (IV) complexes induce rapid tumor cell death in vitro, Int. J. Cancer 116 (2005) 479–486.
Please cite this article as: A. Pantovic, et al., Data supporting the inability of indomethacin to induce autophagy in U251 glioma cells, Data in Brief (2017), http://dx.doi.org/10.1016/j. dib.2017.02.012i