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. 2020 Mar 10;39(18):3754–3773. doi: 10.1038/s41388-020-1251-2

Fig. 2. GPRC5A deficiency promotes NF-kB-mediated ECM1 expression elevation in Lgr5 cells and secreted ECM1-induced AT2 cell enrichment in the S/TB region.

Fig. 2

a Top 10 upregulated pathways in KO:Lgr5 vs WT:Lgr5 cells by KEGG pathway enrichment analysis; b quantitative PCR (qPCR) (top) and western blot (WB) (bottom) of ECM1 in cell lysates (CL) or condition medium (CM) of isolated Lgr5 cells from WT or KO mice; Illustration (c) and WB verification (d), colony formation capacity analysis (e) and tumor initiation capacity analysis (f) of AT2 cells isolated from KO mice cocultured with Lgr5 cells isolated from KO mice treated with an anti-ECM1 neutralizing antibody; g WB analysis of phosphorylated p65 (p-p65), total p65 (t-p65) in Lgr5 cells from WT or KO mice; h qPCR analysis of ECM1 in Lgr5 cells from KO mice (KO:Lgr5) treated with TNF-a, an NF-kB activator, or PS1145, an NF-kB inhibitor; i Luciferase activities of different promoters in KO:Lgr5 cells. ECM1 WT promoter with the potential NF-kB p65 binding site “GGGagatCCC”, ECM1 MT promoter with this site mutated to “TTTagatCCC”; j Luciferase activities tested in KO:Lgr5 cells transiently transfected with ECM1 WT or MT promoters and treated with or without TNF-a (10 ng/ml) for 5 min, PS1145 (20 μM) for 30 min, or PS1145 (20 μM) for 30 min followed by TNF-a (10 ng/ml) for 5 min; k WB analysis of phosphorylated p65 (p-p65), total p65 (t-p65) and ECM1 in KO:Lgr5 cells treated with PS1145, an NF-kB inhibitor; l Flowchart of the isolation and culture of Lgr5 cells from KO mice, knockout of ECM1 or p65, then injected cells through tail vein and harvest the lung tissues for embedding, sectioning and staining; m WB analysis of phosphorylated p65 (p-p65), total p65 (t-p65) and ECM1 in KO:Lgr5 cells after ECM1 or p65 knockdown; n IF analysis of AT2 (SPA) cells in the lung S/TB region after KO:Lgr5 cells with ECM1 or p65 knockdown and injected into NOD/SCID mice through tail vein, (Bar = 100 µm); Data were collected from three independent experiments with triplicate samples. **P < 0.01; ***P < 0.001.