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. 2018 Feb 22;9(3):311. doi: 10.1038/s41419-017-0256-4

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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 Western blot analysis for p-STAT3^Y705, p-STAT5^Y694, p-ERK1/2^T183/Y185, p-AMPK^T172, p-mTOR^S2448, p-4EBP1^T70, p-p70S6K^T421/S424, caspase 3 (total and cleaved) and cleaved PARP1 levels in total cell extracts from HEL and SET2 cells treated with the indicated concentrations of ruxolitinib and/or metformin; membranes were reprobed with the antibody for the detection of the respective total protein or actin, and developed with the SuperSignal™ West Dura Extended Duration Substrate system using a Gel Doc XR+ imaging system. b Gene expression heatmap from qPCR array analysis of HEL cells treated with ruxolitinib (300 nM) and/or metformin (10 mM). mRNA levels are normalized to those of untreated HEL cells and calculated as fold change in expression; genes demonstrating ≥1.5-fold in either direction compared to untreated cells in any treatment are included in the heat map. Two independent experiments of each condition were used for the analysis; green indicates repressed mRNA levels and red elevated mRNA levels. c qPCR analysis of CCND1 and CDKN1B mRNA expression in HEL and SET2 cells treated with ruxolitinib (300 nM) and/or metformin (10 mM) for 48 h. The dashed line represents the mean gene expression in untreated cells and bars represent the fold change in gene expression in HEL and SET2 cells treated with ruxolitinib, metformin, or both compared to their respective untreated cells. The p values and cell lines are indicated in the graphs. *p < 0.05 for metformin-treated and/or ruxolitinib-treated cells vs. untreated cells, #p < 0.05 for metformin-treated or ruxolitinib-treated cells vs. combination treatment at the corresponding doses; ANOVA test and Bonferroni post-test, all pairs were analyzed and statistically significant differences are indicated. d Western blot analysis for p-RB^S807/811levels in total cell extracts from HEL and SET2 cells treated with ruxolitinib and/or metformin; membranes were reprobed with the antibody for the detection of the total RB protein and actin