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. 2021 Apr 21;11:8585. doi: 10.1038/s41598-021-87949-7

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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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Netrin-1 inhibits BMP signaling in MEFs. (a–c) Wild-type MEFs were starved in serum-free medium and then treated with various concentrations of BMP4, BMP6, or BMP9 together with various concentrations of netrin-1 for an hour. Then, BMP signaling was assessed via the analysis of nuclear pSmad1/5 levels. The graphs show the average means with error bars showing the standard deviations of three independent experiments performed in duplicate (one-way ANOVA: ^nsp ≥ 0.05; ^#p < 0.05; ^##p < 0.01; ^###p < 0.001). (d) Wild-type MEFs were treated with various concentrations of netrin-1 at different times before, with, or after the addition of BMP4 [5 ng/ml] to the wells. One hour after the addition of BMP4, BMP signaling was assessed via the analysis of nuclear pSmad1/5 levels. The x-axis shows the time at which netrin-1 was added relative to the addition of BMP4: negative values indicate the addition of netrin-1 before the addition of BMP4, ‘0′ indicates the simultaneous addition of netrin-1 and BMP4, and positive values indicate the addition of netrin-1 after the addition of BMP4. The graph shows the average means with the standard deviations of four independent experiments performed in duplicate (one-way ANOVA: ^####p < 0.0001). (e) Id1 mRNA expression levels in response to BMP4 and netrin-1. MEFs were treated, or not, with 20 ng/ml BMP4 and/or 0.25 μg/ml netrin-1 for x hours followed by analysis of RNA expression levels through real-time qRT-PCR. Shown are the Id1 levels normalized to the Rn18s levels. The graph represents the average means with standard deviations of four independent experiments performed in duplicate (Student’s t-test: **p < 0.01; ***p < 0.001). (f) MEFs of different Ntn1 and Lrig genotypes (Ntn1^+/+ or Ntn1^−/− and Lrig-wild-type or Lrig-null [Lrig1^−/−;Lrig2^−/−;Lrig3^−/−], respectively) were treated with various concentrations of BMP4, and BMP signaling was assessed via analysis of the nuclear pSmad1/5 levels. The graph shows the means with the standard deviations of four Ntn1^+/+;Lrig-wild-type or Ntn1^+/+;Lrig-null biological replicates and three Ntn1^−/−;Lrig-wild-type or Ntn1^−/−;Lrig-null biological replicates determined through three independent experimental repeats. The error bars represent the standard deviations (Student’s t-test: **p < 0.01; ****p < 0.0001. Colors of asterisks represent the respective genotype, according to the graph symbols, compared to Ntn1^+/+;Lrig-WT). (g, h) Cross-titration of netrin-1 and LRIG1 (g) or LRIG3 (h). LRIG1- or LRIG3-inducible Lrig-null and Ntn1^−/− MEFs were treated with different concentrations of doxycycline (between 0 and 1 μg/ml) to induce various levels of LRIG protein expression. BMP signaling was induced using 5 ng/ml BMP4 together with different amounts of netrin-1 (between 0 and 1 μg/ml) for an hour. The fluorescence response for the nuclear pSmad1/5 levels induced by BMP4 is plotted on the y-axis. The doxycycline-induced expression levels of FLAG-tagged LRIG1 or LRIG3 were measured and plotted on the x-axis. Each dot represents the quantification of pSmad1/5 and LRIG-FLAG from each measurement. The graphs show data obtained from three independent experiments performed in duplicate for each concentration of netrin-1 and doxycycline. The R² values for the scattered plots are tabulated in Tables 1 and 2.