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. 2017 Sep 19;7:11893. doi: 10.1038/s41598-017-12131-x

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© The Author(s) 2017

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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CISD2 negatively regulates mRNA expression of antioxidant gene GPX3 and ROS-mediated EGR1 signaling. (A and B) RT-qPCR analysis of expression levels of selected antioxidant genes using shCISD2 transfectant CISD2(−)#1-H1299 (A) or siCISD2 transfected cells harvested at 48 h post transfection (B). (C) GPX3 mRNA expression profile of lung ADC versus normal lung tissues extracted from public domain dataset GSE31210. (D) Scatter plot showing significantly negative correlation between mRNA expression levels of CISD2 and GPX3 in public dataset GSE31210. Correlation test was used to estimate the significance of correlation. (E) Cox’s regression analyses (age adjusted) of GPX3 mRNA expression and prognostic outcome in patients with lung ADC. PFS, progression-free survival; OS, overall survival. (F) EGR1 mRNA expression profile of lung ADC versus normal lung tissues extracted from public domain dataset GSE19188. (G) Scatter plots showing significant negative correlation between mRNA expression levels of CISD2 and EGR1 in public dataset GSE19188. (H) Survival analysis on EGR1 mRNA expression and recurrence-free survival using public domain data GSE31210. Patients were stratified into low EGR1 (solid line) and high EGR1 (dashed line) groups using median EGR1 expression level as cutoff; significance of difference in survival between groups was estimated using log rank test. (I) EGR1 mRNA expression in siCISD2 transfected lung ADC cell lines as measured by RT-qPCR assay. Cells were harvested at 48 h post transfection. (J) Western blot of CISD2 and EGR1 proteins of cells used in (I). (K) Western blot analysis of CISD2 silencing-induced upregulation of EGR1 in CL1-5 cells was reduced when cells were pretreated with 1 mM of NAC for 1 h before siCISD2 transfection. (L and M) Western blotting (L) and RT-qPCR (M) analyses of CISD2 and EGR1 expression in H1299, CL1-0 and CL1-5 cells treated with 100 μM H₂O₂ for 1 h. (N) Western blotting of putative targets downstream EGR1 signaling in CISD2 silencing clones. For (A), (B) (C), (F) and (I), p values were obtained using Student’s t-test, *P < 0.05, **P < 0.01, ***P < 0.001. All data are mean ± SD of at least triplicate measurements.