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. 2018 Jan 29;4:3. doi: 10.1038/s41420-017-0002-9

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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. 3 — a Representative western blot out of three experiments for t-eIF2α and CHOP detected in the lysates of U937 cells pretreated or not for 30 min with GSK (10 μM) and thereafter treated or not for 18 h with TN (3 μM), 4μ8 C (12.5 μM) or both drugs. Each protein was probed with specific antibody followed by peroxidase-conjugated secondary antibodies. β-actin is shown as loading control. The values under each band were obtained using the following formula: (densitometry value of the band under examination / densitometry value of the band of the corresponding β-actin) / (densitometry value of the band under examination in the lysate of untreated cells / the densitometry value of the β-actin band in the lysate of untreated cells). b, c Cell death parameters were evaluated in U937 cells, treated as in a, by calculating PI positive cells as percentage of total cells examined by cytofluorimetry b and subG1 as percentage of the events in the cell cycle examined by cytofluorimetry c. For each parameter ≥ 10.000 events were acquired for each sample. The reported values are the means ± S.D. (N = 5). Statistical analysis by Student’s t-test are shown. d PERK impairment by specific siRNA restores eIF2α expression. U937 treated for 72 h with equal amount of scrambled siRNA or PERK specific siRNA were treated or not with 4μ8 C (12.5 μM) + TN (3 μM) for 18 h. PERK, t-eIF2α and CHOP were analyzed by western blot in the cell lysates by specific antibodies followed by peroxidase-conjugated antibodies. β-actin is shown as loading control and to confirm the specificity of the transfected siRNA. The values under each band were obtained using the formula: (densitometry value of the band under investigation / densitometry value of the band of the corresponding β-actin) / (densitometry value of the band under investigation in the lysate of scrambled treated cells / the densitometry value of the β-actin band in the lysate of scrambled treated cells). This type of experiment was performed twice with comparable results. e, f Death parameters were evaluated in U937 cells, treated as in d, by calculating PI positive cells as percentage of total cells examined by cytofluorimetry e and subG1 as percentage of the events in the cell cycle examined by cytofluorimetry f. For each parameter ≥ 10.000 events were acquired for each sample. The reported values are the means ± S.D. (N = 3). Statistical analysis by Student’s t-test are shown