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. 2021 Jun 12;12(6):610. doi: 10.1038/s41419-021-03897-0

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© The Author(s) 2021

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. 6 — A Kaplan–Meier analysis of patients overall survival data based on high versus low expression of KLF9 in glioma, grades II–IV, from the TCGA dataset. B, C U87MG or U251 cells were transfected with control/KLF9-overexpressing vectors for 48 h. Cells were harvested for CCK-8 assay measuring the viable cell numbers (B) or flow cytometry analysis to measure intracellular ROS level (C). D U87MG or U251 cells were transfected with control or KLF9-overexpressing vectors, and then harvested for western blotting after treated with vehicle or N-acetyl-l-cysteine (5 mM) for 48 h. Densitometric quantification of cleaved-caspase3/caspase3 ratio from at least three independent assays was indicated on top of each band, respectively. E Real-time PCR analysis to assess the mRNA levels of the oxidative stress-related genes in U87MG cells transfected with control or KLF9-overexpressing vectors for 48 h. F HEK293T cells were co-transfected with SOD1 promoter-firefly luciferase, renilla luciferase, and KLF9-overexpressing vectors for 48 h. Cells were then harvested for dual-luciferase reporter assay following the manufacturer’s instructions. G Control, U87/U251-GPR17, or U87/U251-shGPR17 cells were prepared as in Fig. 2. Cells were harvested for real-time PCR analysis to assess the mRNA levels of SOD1. H Control or U87/U251-GPR17 cells were transfected with scramble or KLF9-knockdown vectors, together with SOD1 promoter-firefly luciferase, Renilla luciferase constructs for 48 h. Cells were then harvested for dual-luciferase reporter assay following the manufacturer’s instructions. I Control or U87/U251-shGPR17 cells were transfected with control or KLF9-overexpressing vector for 48 h. Flow cytometry analysis was performed to assess mitochondrial ROS levels. J, K Control or U87/U251-GPR17 cells were transfected with scramble or KLF9 shRNAs for 48 h. Cells were then harvested for CCK-8 assays (J) or western blot against cleaved-caspase3 (K). Densitometric quantification of the cleaved-caspase3/caspase3 ratio from at least three independent assays was indicated on top of each band, respectively. For all panels, data represent the means ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t-test.