Fig. 3.

Location-specific transcriptional signatures of mLN FSCs are imprinted by microbiota. a CD45⁻Ter119⁻CD31⁻gp38⁺ FSCs were isolated from mLNs and pLNs of GF or SPF mice, and RNA-seq and subsequent analysis was performed. DEGs were identified in colonization-dependent (SPF vs. GF) and location-dependent (mLNs vs. pLNs) pairwise comparisons (|log₂(FC)| ≥ 1 and q-value ≤ 0.05). Heatmap represents 1411 DEGs. Mean-centered log₂(RPKM) values are depicted. Data pooled from two independent experiments. b CD45⁻CD24⁻CD31⁻gp38⁺ FSCs were isolated from endogenous mLN-SPF and pLN-SPF, transplanted pLN-SPF and mLN-SPF or transplanted mLN-GF. RNA-seq^L and subsequent analysis was performed. Colored numbers in scatterplots represent DEGs (|log₂(FC)| ≥ 1 and q-value ≤ 0.05) for the pair-wise comparisons of FSCs. On the x-axis log₂(FC) of gene expression from FSCs (endogenous LNs) is plotted. (left) On the y-axis log₂(FC) of gene expression from FSCs from transplanted mLN-SPF vs. pLN-SPF is plotted. (right) On the y-axis log₂(FC) of gene expression from FSCs from transplanted mLN-GF vs. pLN-SPF is plotted. Data pooled from two to four independent experiments (n = 3–8). c GO analysis of biological processes of genes persistently up-regulated (Maintained, blue dot) or down-regulated (Repressed, pink dot) in FSCs of transplanted mLN-SPF. d Heatmaps of expression of genes involved in RA metabolic process or of soluble mediators within FSCs from indicated groups. Numbers in brackets indicate average log₂(RPKM) expression of respective genes across all experimental groups. Data pooled from two to four independent experiments (n = 3–8). DEG differentially expressed genes, AA amino acid, FC fold-change, FSC fibroblastic stromal cells, GO gene ontology, RA retinoic acid, reg. regulation, res. response, RPKM reads per kilobase of exon length per million mapped reads, sig. signaling