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. 2020 Feb 7;39(14):2948–2960. doi: 10.1038/s41388-020-1196-5

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© The Author(s) 2020

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. 2 — a Lysophosphatidylinositol (LPI) 72 h after ACSL3 knockdown in A549 cells. Cells were transduced with either an empty vector control (pLKO) or an shRNA against ACSL3 (#1), 72 h later lipids were extracted and analyzed by mass spectrometry-based shotgun lipidomics n = 4/group. Data are presented as mean ± SD. b Schematic model for LPIAT1 function in phospholipid remodeling. c PGE2 ELISA assay for A549, A427, H1264, and H358 cell lines. Cells were transduced with either an empty vector control (pLKO) or shRNA against LPIAT1 (shLPIAT1 #1). Cells were transduced, selected with puromycin and plated for PGE2 measurement. 24 h later the media was changed, incubated for additional 24 h and PGE2 production was quantified from the supernatant n = 3/group. Data are presented as mean ± SD. d Relative cell number of A549, A427, H1264, and H358 cell lines transduced with an empty vector control (pLKO) or three different shRNAs against LPIAT1. n = 3/group. Data are presented as mean ± SD. e Relative cell number of A549 cell line transduced with either pLKO.1 hygro + pLenti-GIII-CMV-GFP-2A-Puro (control) or pLKO.1 hygro-shACSL3 #2 + pLenti-GIII-CMV-GFP-2A-Puro (shACSL3) or pLKO.1 hygro + pLenti-GIII-CMV-GFP-2A-Puro-LPIAT1 (LPIAT1 overexpression) or pLKO.1 hygro-shACSL3 #2 + pLenti-GIII-CMV-GFP-2A-Puro-LPIAT1 (shACSL3 + LPIAT1 overexpression) n = 3/group; o/e: overexpression. Data are presented as mean ± SD. f PGE2 quantification from A549 cell line supernatants. Cells were transduced with either pLKO.1 hygro + pLenti-GIII-CMV-GFP-2A-Puro (control) or pLKO.1 hygro-shACSL3 #2 + pLenti-GIII-CMV-GFP-2A-Puro (shACSL3) or pLKO.1 hygro + pLenti-GIII-CMV-GFP-2A-Puro-LPIAT1 (LPIAT1 overexpression) or pLKO.1 hygro-shACSL3 #2 + pLenti-GIII-CMV-GFP-2A-Puro-LPIAT1 (shACSL3 + LPIAT1 overexpression). Cells were transduced, selected with 2 μg/mL puromycin or 150 ng/mL hygromycin and plated for PGE2 measurement. 24 h later the media was changed, incubated for additional 24 h and Supernatants were analyzed by ultra-high performance liquid chromatography with tandem mass spectrometry n = 4/group. Data are presented as mean ± SD. g Representative images of soft agar colony formation assay in A549 cells transduced as in f. h Histograms showing quantification of colony number and size of A549 cells grown in soft agar treated as in f. n = 3/group. AU arbitrary units. Data are presented as mean ± SD. Statistical analyses were done using two-tailed unpaired Student’s t test or one-way ANOVA. *p < 0.05, **p < 0.01, ****p < 0.0001.