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. 2018 May 21;26(2):276–290. doi: 10.1038/s41418-018-0118-3

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — RPE-1 control cells were exposed to MitoTam (2.5 μM) for 48 h in high (4.5 g/L) and low (1 g/L) glucose medium (a) or in the presence of 2-deoxyglucose (2-DG; 25 mM) (b), and cell survival was evaluated by annexin V/Hoechst negativity using flow cytometry. c RPE-1 control and senescent cells (BrdU) were exposed to MitoTam (2.5 μM) for 48 h and lactate level was assessed. d Expression of ANT1, ANT2 and ANT3 transcripts in RPE-1 control and senescent (BrdU) cells were detected by qRT-PCR. e Control and senescent (BrdU) RPE-1 cells, as well as BJ control (pd 31) and senescent (pd 82) cells were assessed for the level of the ANT2 protein by western blotting. β-actin was used as loading control. f RPE-1 control cells were exposed to MitoTam (2.5 μM) for 48 h after downregulation of ANT2 using specific siRNAs, and their survival was evaluated by annexin V/Hoechst negativity using flow cytometry. g RPE-1 control and senescent (BrdU) cells, as well as their ANT2-overexpressing counterparts, were exposed to MitoTam (2.5 μM) for 48 h and viability was evaluated as annexin V/Hoechst negativity by flow cytometry. h Structure of mitochondria in RPE-1 control and senescent (BrdU) cells as well as their ANT2-overexpressing counterparts exposed to MitoTam (2.5 μM) for 24 h were assessed for mitochondrial morphology using transmission electron microscope. Scale bar represents 1 μm. i RPE-1 control and senescent (BrdU) cells were exposed to oligomycin A (5 μM) for 48 h and their survival was evaluated by annexin V/Hoechst negativity using flow cytometry. j Control and senescent (BrdU) RPE-1 cells were exposed to MitoTam (2.5 μM), MitoVES (2.5 μM) or rotenone (0.5 μM) for 24 h, and the cells were assessed for ΔΨ_m,i using TMRM and flow cytometry. k RPE-1 control and senescent (BrdU) cells were exposed to rotenone (0.5 μM) in combination with CCCP (10 μM) for 48 h and their survival was evaluated by annexin V/Hoechst negativity using flow cytometry. Data in all graphs represent means ± S.D. from three independent experiments. The asterisk represents p < 0.05