Fig 2: Validation of three novel ZFP36 target genes involved in EMT(Panel A) Boxplot of Log 2 expression values of MACC1, SOX9 and ZEB1 in 23 normal colon mucosa (Normal), 30 primary colon carcinoma (CRC) and 27 liver metastases (Mts) samples. The thick line indicates the median value, the coloured box indicates the interquartile range and the whiskers the minimum and maximum values excluded outliers. Open circles represent data points outside the whiskers. (Panel B) Schematic representation of the 3'UTRs sequences of MACC1, SOX9 and ZEB1. A-U rich sequences (ARE) are highlighted in bold. (Panel C) HCT116 and SW480 cells were transfected with an empty vector (pCDNA3.1) or a ZFP36-overexpressing vector (pCDNA3.1-ZFP36). RNA was extracted after 48 hours and MACC1, SOX9, ZEB1 mRNA levels were analysed through qRT-PCR analysis. Results are represented as means of three experiments (+/-SEM) and GAPDH was used as endogenous control. *p < 0.05. (Panel D) HCT116 and SW480 were infected with an empty vector (pRRL) or a ZFP36-overexpressing vector (ZFP36) and corresponding total protein lysates were analysed through Western blotting techniques with antibodies against ZEB1, MACC1, SOX9 and ZFP36. Actin was used as loading control. (Panel E) A fragment of the 3'UTRs of MACC1, SOX9 and ZEB1 was cloned in a pGL3 vector, downstream of the Luciferase gene. These constructs where co-transfected with a ?-gal reporter plasmid and with an empty vector (pCDNA3.1) or ZFP36 overexpressing vector (pCDNA3.1-ZFP36) in HEK293T cells. Cells were harvested after 48 hours, luciferase activity was measured and normalized over ?-gal signals. Results are represented as means of three independent experiments +/-SEM. *p < 0.05, **p < 0.001, ***p < 0.0001.

Fig 3: Single-cell RNA-sequencing analysis of human healthy control chondrocyte tissue samples.A Seven healthy human chondrocyte clusters. Visualisation of clustering by t-SNE plot of healthy control samples, identified by cell type. B Monocle pseudotime trajectory showing the progression of seven clusters of healthy chondrocytes. C Heat map of scaled gene expression data for the top 10 differentially expressed genes identifying each cluster, with selected genes listed. D Gene expression plots demonstrating high expression of SH3BGRL3 in SH3BGRL3hi fibrocartilage chondrocytes-1, IGFBP5 in IGFBP5hi fibrocartilage chondrocytes-2, CDC20 in CDC20hi cartilage progenitor cells-1, KIAA0101 in KIAA0101hi cartilage progenitor cells-2, EIF5A in EIF5Ahi regulatory chondrocytes, SOX9 in SOX9hi prehypertrophic chondrocytes and TXNIP in TXNIPhi homeostatic chondrocytes. Dot colour corresponds to the level of gene expression in each cell. t-SNE, t-distributed stochastic neighbour embedding. E Representative immunohistochemistry staining of SH3BGRL3, IGFBP5, BIRC5, SOX9, EIF5A and CDC20 in healthy cartilage tissues. Scale bar, left, 500 µm; right, 50 µm, and quantification of positive cells of different areas (superficial, middle, deep) in cartilage tissues displayed by box plot (n = 5). *p < 0.05.

Fig 4: Smad and NF-?B signaling pathways play a synergistic role in HO formation, and palovarotene inhibits HO formation by blocking the Smad and NF-?B signaling pathways. (a) The mRNA expression of OCN, SOX9, RUNX2, Smad1, Smad5, and ID1. Data are means ± SD (n = 3). *p < 0.05 and **p < 0.01. #p < 0.05 and ##p < 0.01. (b) Immunofluorescence against ID1 (n = 3). Scale bar = 25 µm. (c) Western blot analysis and relevant quantitative analysis of p-p65 and ID1. Data are means ± SD (n = 3) *p < 0.05 and **p < 0.01. #p < 0.05 and ##p < 0.01. (d) Immunofluorescence against p65 (n = 3). Scale bar = 25 µm. (e) Western blot analysis and relevant quantitative analysis of p65 and immunofluorescence against P65 in the previous coculture model series experiments (n = 3). Data are means ± SD (n = 3). *p < 0.05 and **p < 0.01. #p < 0.05 and ##p < 0.01. Scale bar = 25 µm.

Fig 5: The inflammatory microenvironment promotes osteogenic differentiation of TSCs, and palovarotene can influence osteogenesis. (a) CCK8 proliferation assay was used to determine the dose range of palovarotene. Palovarotene at 0.5, 1, and 2 µM concentration had less effect on TSCs proliferation. Coculture system of activated macrophages and TSCs was constructed. (b) ALP and ARS staining in TSCs after coculture with macrophages. Data are means ± SD (n = 3). *p < 0.05 and **p < 0.01. #p < 0.05 and ##p < 0.01. Scale bar = 50 µm. (c) Western blot analysis and relevant quantitative analysis of OCN, SOX9, and RUNX2. Data are means ± SD (n = 3). *p < 0.05 and **p < 0.01. #p < 0.05 and ##p < 0.01. (d) The mRNA expression of OCN, SOX9, RUNX2, MMP-9, IL-1ß, IFN-?, and TNF-a. Data are means ± SD (n = 3). *p < 0.05 and **p < 0.01. #p < 0.05 and ##p < 0.01.