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. 2021 May 18;11:10495. doi: 10.1038/s41598-021-89860-7

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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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Expression of IL-4/IL-13-associated genes requires STAT3 availability. Whole spleen homogenate derived from naïve 6–8-week-old female BALB/c mice were pre-incubated with STAT3i or STAT6i in vitro for 3 h prior to stimulation with 50 ng/mL IL-4 for 24 h. Fluidigm Biomark 48.48 analyses were performed on single CD3⁺CD4⁺CD8^-IL-4R⍺⁺ cells, evaluating transcript expression of genes-of-interest. (a) dichotimised gene expression profiles of genes-of-interest between treatments. Statistical significance was determined by performing a Fisher’s exact test. (b) Co-expression analysis of members of the STAT family (STAT6, STAT3 and STAT5a). (c) PCA was conducted to evaluate heterogeneity in transcriptional profiles, particularly whether the heterogeneity can be explained by the treatment (refer Sup. Fig. 7a,b for loadings). (d) For genes expressed by at least 15% of cells, Spearman’s rank correlation analysis was performed to evaluate correspondence of genes pairwise. According to the similarity in transcriptional profiles, a major cluster of positively correlated genes were identified and highlighted, along with a negatively correlated cluster (refer Sup. Fig. 7a for dendrogram). The experiment was repeated twice, pooling the data. n ~ 15 cells per group.