Fig. 7

In vitro studies and SCENIC analysis identified crucial regulators of MMT. (a) mRNA levels of selected markers (fibro/osteoblastic markers and ECM related proteins). Data was normalized as log2(FC + 1). FC: fold change to control. (b) Representative western blot images of fibro/osteoblastic markers and ECM related proteins. MRC1⁺Macs were incubated in OM for 7 days. (n = 5,5 for GM, OM) (c) Representative images of immunofluorescence after MRC1⁺Mac incubated in OM for 3 or 7 days. Anti-MRC1: green, anti-α-SMA: red, DAPI: blue. Scale bar: 200 μm. (d) Heatmap of the mean value of area under the recovery curve (Aucell) value of expression regulation by the transcription factors, as estimated using SCENIC. The top 10 transcription factors genes (TFs) of each subcluster were shown. (e) The expression dynamics of top 10 TFs in MDSC. GATA5, MKX, ZNF853 were hardly detected in transcriptome. (f) RT-qPCR examined AR, FOXC1 and NFIB mRNA levels changes after OM incubation of MRC1⁺Mac. (n = 4 for each group) (g) The protein levels of FOXC1 in MRC1⁺Mac after OM incubation. (h) GSEA plot showing PI3K-AKT signaling pathway is significantly activated in MDSC. (i) The phosphorylation levels of AKT upregulated in vitro after OM induction. (j) The proteins levels of myofibro/osteoblastic markers and PI3K-AKT pathway after treatments of OM an si-FOXC1 transfection. (k) Representative images of alizarin red staining of each group. (l) The quantitative results of alizarin red staining (n = 6)