Fig. 4: TGFβ1 secreted by MKs activates LepR⁺ SSCs via the Smad2/Slc39a14 signaling pathway.

a RNA-seq analysis revealed changes in gene expression in LepR⁺ SSCs co-cultured with MKs (n = 3 each). b qPCR analysis of the expressions of Smad2 and Slc39a14 in LepR⁺ SSCs, with or without MKs, from WT mice (n = 6 per group). c Western blotting analysis of the expression of Smad2 and Slc39a14 in LepR⁺ SSCs, with or without MKs, from WT mice (n = 3 per group). d Representative immunostaining images of Smad2 (red) and Slc39a14 (green) in LepR⁺ SSCs, with or without MKs, from the BM of TGFβ1^MKΔ/Δ and TGFβ1^fl/fl mice (n = 6 per group). Scale bar, 100 µm. e Colocalization of Smad2 (red) with Slc39a14 (green) in LepR⁺ SSCs, with or without MKs, from the BM of TGFβ1^MKΔ/Δ and TGFβ1^fl/fl mice (n = 6 per group). Ctrl, control. f A schematic representation of the neural network model of Smad2 binding to the promoter region of Slc39a14, predicted by AlphaFold 3. g A plot of the predicted aligned error of the complex predicted by AlphaFold 3 (pTM + ipTM = 0.91). h A plot of the binding site and amino acid residues of Smad2–Slc39a14 analyzed by PyMol. i Dual-luciferase assays of 293T cotransfected with WT or mutated Slc39a14 (LUC), combined with pcDNA3.1-Smad2 or pcDNA3.1 vetor. j ChIP assay of Smad2 binding to Slc39a14 promoters in LepR⁺ SSCs transfected with pcDNA3.1-Smad2 or pcDNA3.1. Immunoprecipitated DNA and the input DNA were detected by PCR. Primer sequences were designed for Slc39a14 promoter regions located in the promoter region of the Slc39a14 gene, with IgG as a negative control. Data on graphs are shown as mean ± SD. An unpaired two-tailed t-test was used to analyze the data in b and i. *P < 0.05, **P < 0.01 and ***P < 0.001. For all panels in this figure, data are representative of three independent experiments.