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. 2019 Oct 14;10(10):779. doi: 10.1038/s41419-019-2024-0

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© The Author(s) 2019

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. 4 — a Monitoring of Δψm loss in CBD-treated leukemic cells. Jurkat cells were double-stained with MtGreen and TMRE, mitochondria (ROI) were selected (upper panel) and intensity of fluorescence was monitored by confocal microscopy after CBD (30 μM) administration (lower panel, n = 36; see also Supplementary movie 1). b Intensity of TMRE fluorescence as an indicator of Δψm in Jurkat cells, treated with different concentrations of CBD (0–100 μM) during 10 min. FCCP (10 μM) was used as a positive control. Data are mean ± SD (n = 8 in three independent experiments). Statistical comparison with control was performed by means of one-way ANOVA test (*p < 0.05; ****p < 0.0001). c EYFP-Cyt-C is localized in mitochondria of Jurkat cells after 12 h of transfection. EYFP-Cyt-C and TMRE fluorescence are colocalized (200 nM TMRE, 200 ng EYFP-Cyt-C, pseudocolor). Images were acquired by confocal microscopy at 12 h after transfection, scale bar: 10 μm. d Representative images of five Jurkat cells, transfected with EYFP-Cyt-C (as in c). Dense green fluorescent puncta (pseudocolor) reflect Cyt-C localization in intact mitochondria. Scale bar: 10 μm. e Representative images of five Jurkat cells, transfected with EYFP-Cyt-C (as in c) and treated with CBD (30 μM, 1 h). Cyt-C was released from mitochondria as evidenced by a diffuse EYFP-CytC distribution. Scale bar: 10 μm. f EYFP-Cyt-C distribution in Jurkat cells, expressing EYFP-Cyt-C, at 0, 5, and 20 min with CBD (30 μM). Scale bar: 10 μm. g Caspase-9 and caspase-3 activity in vehicle- and CBD- treated (30 μM, 12 h) Jurkat cells. Staurosporine (STS, 5 μM) was used as a positive control. After incubation, cells were lysed, and caspase activity was determined by colorimetric assay (BioVision). Fold increase in activity compared to control was plotted as mean ± SE (four independent experiments). One-way ANOVA test was performed to compare CBD-treated to control group (*p < 0.05; **p < 0.01). h Pretreatment with mPTP inhibitor CsA (10 μM) prevents Cyt-C release, induced by CBD (30 μM; 1 h). Compare these images with Fig. 4d, e (vehicle- and CBD-treated cells) and note the protective effect of CsA. Scale bar: 10 μm. i ROS production as evaluated by fluorescent microscopy in DCF-loaded (2 μM) Jurkat cells. CBD effect on ROS production was monitored after 1 h of incubation. PMA (1 μM) was used as a positive control