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. 2021 Apr 6;10(4):32. doi: 10.1038/s41389-021-00321-2

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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. 4 — A IL-6 mRNA levels measured by RT-PCR (left), 72h-time course of released IL-6 protein levels measured upon P/I (50 µg/ml/1 μM) (middle) or TNF (10 ng/ml) (right) stimulation. Data represent means ± SD of triplicates for two clones of each cell line (N = 3). Statistical analyses were carried out using GraphPad Prism software. Differences among groups were assessed using one-tailed Student’s t tests. P < 0.05 was considered to be statistically significant. B Upper panel, Volcano plot of differential mRNA expression in A549-WT and KI cells; microarray probe sets with an absolute log2 fold change of ≥ 1 and a false discovery rate-adjusted p ≤ 0.01 are colored in blue. Lower panel, Gene set enrichment analysis of the data shown in the upper panel. Shown are the top 50 of 69 gene sets significant at a false discovery rate < 0.05. NES Normalized Enrichment Score. Highly significant pathways were (i) cancer (p53, KRAS) (ii) inflammation (NF-κB, TNF), (iii) cancer-related processes (extracellular matrix (ECM), apoptosis, and angiogenesis), with a particular enrichment of gene sets linked to ECM regulation (matrisome, ECM organization, proteoglycans, cell adhesion-matrix glycogonjugates, and ECM proteoglycans). C Relative quantitative PCR measurement of eight selected mRNAs contributing to ECM regulation signatures at basal level in A549-WT or KI: Versican (VCAN), Fibroblast growth factor 5 (FGF5), Fibronectin Leucine Rich Transmembrane Protein 2 (FLRT2), CEA Cell Adhesion Molecule 6 (CEACAM6), Interleukin-6 (IL-6), and fibrinogen genes FGA, FGB, and FGG). Means ± SD of triplicates for two clones of each cell line are shown (N = 2). Statistical analyses were carried out as described in (A). Microarray values for the eight leading edge mRNAs (Fig. 4C) are shown in Supplementary Fig. 8. D Upper panel, Box plot of IL-6 protein levels as measured by SomaScan (SOMAmer anti_2573_20) in A549-WT and KI cells, at baseline and after a 24 h stimulation with P/I. Lower panel, Gene set enrichment analysis of longitudinal SomaScan data comparing the effects over time in A549-WT and KI cells. A selection of 124 gene sets related to ECM regulation was used. Shown are the 13 gene sets significant at a false discovery rate < 0.05; NES Normalized Enrichment Score.