Fig 2: RUNX2 gene and protein expression analysis in the L88/5 cell line by immunocytochemistry. (A) RUNX2 expression by rhodamine immunostaining (red) in L88/5 (36 hours after pCMV6-AC-GFP TRPS1 plasmids transfection) and nuclear counterstaining by DAPI (blue). The graph resumes the staining quantification by ImageJ tool. Scale bars = 50 µm. RUNX2 immunocytochemistry values in mutants were statistically significant, but the one for the c.343C>T variant (p < 0.001, Student's t test). (B) Gene expression analysis of RUNX2 in wild-type and pCMV6-AC-GFP TRPS1 transfected L88/5 cells. The y-axis reports the binary logarithm (ln2) RQ in mRNA expression versus mock, used as calibrator reference. Statistical analysis: ordinary one-way ANOVA. RUNX2: F = 220.9, p = <0.0001; multiple comparisons p values: wt = <0.0001, c.47G>A = 0.0368, c.203A>C = 0.0846, c.343C>T* = <0.0001, c.422C>T* = 0.0011, c.2894G>A = <0.0001. RQ = relative fold expression change.

Fig 3: Proteomic differences of common SCCs and rare SCCs.a Principal-component analysis (PCA) of common SCCs and rare SCCs. b The association of common and rare SCCs with 9 variables (two-sided Fisher’s exact test was used for categorical variables and two-sided Wilcoxon rank-sum test was used for continuous variables), and the heatmap of significantly DEPs (Wilcoxon rank-sum test, BH-adjusted p < 0.05, fold change > 2) in common SCCs and rare SCCs. c Enriched pathways of significantly DEPs (two-sided Wilcoxon rank-sum test, BH-adjusted p < 0.05, fold change > 2) in common SCCs and rare SCCs. d Proteins in pathways that were differentially expressed in common SCCs and rare SCCs, and representative DEPs. e The PLIN1 protein expression in 17 SCCs. f Representative PLIN1 fluorescence in situ hybridization signal patterns (red signals = PLIN1, green signals = CEP15), left, this case (Anus_3) was scored negative for PLIN1 amplification. PLIN1/nucleus ratio = 2.52; right, this case (Anus_10) was scored as positive for PLIN1 amplification. PLIN1/nucleus ratio = 6.2. The boxes indicate the interquartile ranges, and no outliers are shown. g Differential expressed TFs (two-sided Wilcoxon rank-sum test, BH-adjusted p < 0.05, fold change > 2) in common SCCs and rare SCCs. The two TFs, RUNX2 and FOXO1, were labeled in blue. h The expression heatmap of downstream transcriptional targeted genes (TGs) regulated by RUNX2 and FOXO1 (in bold). The expression level was scaled by row. i A scatterplot showed the association between the protein abundance of RUNX2 (x-axis) and FOXO1 (y-axis). Pairwise Spearman correlation. j Immunohistochemistry staining for RUNX2, FOXO1, and PLIN1 expression in rare SCCs (one case of thyroid SCC and one case of pancreatic SCC) was concordant with the mass spectrometry findings. Scale bar, 100 µm. k Diagram depicted our hypothesis of lipid metabolism upregulation contributing to rare SCC aggressiveness and metastasis. Source data are provided as a Source Data file.

Fig 4: Enhanced ß-catenin decreases SMAD1, and together they mediate SB216763-induced osteoblastic-endothelial transition.(A) Immunoblotting of SMAD1, phosphorylated SMAD1 (p-SMAD1), and ß-catenin from osteoblasts treated with different doses of SB216763 (n = 6). (B) Immunoblotting of SMAD1 from osteoblasts transfected with ß-catenin siRNA together with 10 µM SB216763 (n = 6). SCR, scrambled siRNA. (C) Immunoblotting of ß-catenin, SMAD1, Cbfa1, osterix (OSX), Flk1, and VE-cadherin (VE-cad) from osteoblasts with overexpression of ß-catenin or knockdown of SMAD1 or both (n = 6). ß-Actin was used as control. CMV–ß-catenin, CMV promoter–driven ß-catenin cDNA. (D) Micro-CT images of ectopic bone formation and relative volume of bone formation in the implants after cell transplantation. Osteoblasts were used as controls. SB216763, SB216763-treated osteoblasts. SB216763/CMV-SMAD1, SB216763-treated osteoblasts infected with lentiviral vectors containing CMV promoter–driven SMAD1 cDNA. SB216763/ß-catenin si, SB216763-treated osteoblasts infected with lentiviral vectors containing ß-catenin siRNA (si) (n = 6). Scale bar: 5 mm. (E) Laser Doppler perfusion images and percentage of blood flow perfusion after cell transplantation (n = 8). Osteoblast and mouse pulmonary endothelial cells (ECs) were used as controls. Scale bar: 10 mm. D and E were analyzed for statistical significance by ANOVA with post hoc Tukey’s analysis. The bounds of the boxes are upper and lower quartiles. The line in the box is the median, and the whiskers are the maximum and minimal values. Data are shown as mean ± SD.

Fig 5: In postnatal development, Meflin+ cells differentiate into bone chondrocytes and osteocytes and PDGFRa-positive stromal cells in BM. (A) tdTomato IHC showed Meflin+ immature chondroblast-like cells in the articular cartilage (a') and fibroblastoid cells in the periosteum (PO) (a?) of the femoral epiphysis at P4 (arrows). Those cells give rise to chondrocytes and osteoblasts that line the surfaces of the trabecular bone at P21 and P49 (red arrowheads). tdTomato+ Meflin lineage cells include mature osteocytes (green arrowheads). Scale bars, 200 µm. (B) Meflin+ cells (red) are positive for Sox9 (green) at P4 (yellow arrowheads), whereas Meflin lineage cells are either positive (yellow arrowheads) or negative (white arrowheads) for Sox9 at P21 and P49, suggesting the tdTomato+ cells include cells in different stages of chondroblast maturation. Boxed regions are magnified in lower panels. Scale bars, 100 µm. (C) Meflin+ cells at P4 and Meflin lineage cells at P21 and P49 (red) are positive for Runx2 (green) in the femoral epiphysis and diaphysis (arrowheads). Note that most Meflin lineage cells that line the surface of the bone are positive for Runx2 (green arrowheads), whereas there are some Runx2-negative Meflin lineage cells in the periosteum (lower panels, arrows) at P21 and P49. Dashed lines indicate the area of the femoral diaphysis at P4. SM, skeletal muscle. Scale bars, 100 µm. (D) Meflin+ cells at P4 and Meflin lineage cells at P21 and P49 (red) were detected in the perisinusoidal region. They were positive for PDGFRa (green) in the BM in the postnatal development (arrows)