Fig 2: The effect of microtubule (MT) stabilization in the chondrogenesis of SMSCs. (A) Western blot analysis of Ace-Tubulin, Col I, Col II, SOX9, SMAD3, and phosphorylated SMAD3 expression in synovial mesenchymal stem cells (SMSCs) treated with nocodazole (N) and docetaxel (D) in chondrogenic medium (CM) for 1 week. (B) Quantification of the data of (A). n = 5. (C) Immunofluorescence staining of Ace-Tubulin and F-actin. Scale bar, 50 μm. The docetaxel treatment increased the expression level of Ace-Tubulin. (D) Co-immunofluorescence staining of Col I and Col II. Scale bar, 100 μm. (E) Safranin O staining (SO) and Alcian blue (AB) staining of SMSC micro-mass culture grown in chondrogenic medium (CM) treated with nocodazole (N) and docetaxel (D) for 4 weeks. (F) pellet cultures of SMSCs in chondrogenic medium (CM) treated with nocodazole and docetaxel for 4 weeks. SO staining, toluidine blue (TB) staining, and immunohistochemical staining of Col II and Col I were performed to access the chondrogenesis of SMSC pellets. Scale bar, 100 μm. Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 3: Effect of MLT+Ptero on apoptosis-related proteins in colon cancer cells. HCT116, SW480, and HT29 colon cancer cells were exposed to MLT (0.5, 1 mM) and/or Ptero (20, 40 µM) for 24 h and then were subjected to Western blotting for PARP, cleaved caspse-3, Bcl-xL, NEDD9, SOX10, SOX9, and β-actin.

Fig 4: Immunostaining of alveolar differentiation markers in the lungs of HRAS-KO and/or NRAS-KO mice.a Representative images of immunostaining for Ricinus communis agglutinin-I (RCA-I, AT1 lineage, green) and Surfactant protein-C (SftpC, AT2 lineage, red) in paraffin sections of the lungs of newborn P0 mice of the indicated genotypes. Regions of SftpC+ cell accumulations are marked by tailed arrows. Scale bar: 75 µm. The bottom bar graphs quantify, respectively, the average fluorescence intensity (F.I.) of the RCA-I immunoassay signals (relative to Control animals), and the percentage of SftpC+ pneumocytes (relative to total nuclei) in the lungs of P0 individuals of the indicated genotypes. Data expressed as the mean ± s.e.m. Ten separate microscopic fields were quantified for each individual analyzed in each genotype, n = 3 for all genotypes in RCA-I quantification, and n = 3 for Control and n = 4 for the rest of genotypes in SftpC quantification. *p < 0.05, **p < 0.01. b Representative images of immunostaining for Surfactant protein-C (SftpC, AT2 lineage, red) and Ricinus communis agglutinin-I (RCA-I, AT1 lineage, green) in paraffin sections of the lungs of newborn P0 and E18.5 mice of the indicated genotypes. Distal-tip like alveolar structures are marked by arrow heads. Co-immunolabeled, alveolar bi-potent progenitor cells are marked by tailed arrows. Scale bar: 75 µm and 25 µm in the magnified boxed areas. The bottom bar graphs quantify the percentage of alveolar bi-potent cells (RCA-I+/SftpC+) relative to total number of SftpC+ cells in the lungs of P0 or E18.5 individuals of the indicated genotypes. Data expressed as the mean ± s.e.m. Ten separate microscopic fields were quantified for each individual analyzed in each genotype. n = 3 for all genotypes of E18.5 lungs, n = 3 for Controls and n = 4 for the rest of genotypes in P0 lungs. *p < 0.05, **p < 0.01, ***p < 0.001. c Representative images of immunostainings for Sox9 (distal-tip progenitors, red) in peripheral or inner parenchymal areas of E18.5 lungs from the indicated genotypes. Scale bar: 50 µm and 10 µm in the magnified boxed areas. The bottom bar graphs quantify the percentage of Sox9+ cells relative to total number of cells. Data expressed as the mean ± s.e.m. Non-specific erythrocyte staining indicated by arrow heads. Ten separate microscopic fields were quantified for each of the three individuals analyzed in each genotype. n = 3 individuals for all genotypes. *p < 0.05, **p < 0.01, ***p < 0.001

Fig 5: Characterization and trilineage differential potency of SMSCs from osteoarthritis (OA) keen synovium. (A) Surface marker expression of synovial mesenchymal stem cells (SMSCs). Results showed negativity for hematopoietic marker (CD34) and macrophage marker (CD68) and showed positivity for mesenchymal stem cell (MSC) markers like CD105 and CD90. (B) Multi-directional differentiation of SMSCs; chondrogenesis was accessed by safranin O staining (SO), osteogenesis was accessed by alizarin red S staining (ARS), and adipogenesis was accessed by Oil red O staining. (C) The EdU staining of SMSCs after the treatment of different concentrations of nocodazole (N) (5, 10, 25, 50, 100 nM) and docetaxel (D) (0.5, 1, 2.5, 5, 10 nM) in chondrogenic medium (CM) for 1 week. Scale bar, 100 μm. (D) Quantification of the data of (C). n = 5. (E) The cell viability of SMSCs after the treatment of different concentrations of nocodazole (N) (5, 10, 25, 50, 100 nM) and docetaxel (D) (0.5, 1, 2.5, 5, 10 nM) in chondrogenic medium (CM) for 1 week. (F) Crystal violet staining of SMSCs after the treatment of different concentrations of nocodazole (N) (5, 10, 25, 50, 100 nM) and docetaxel (D) (0.5, 1, 2.5, 5, 10 nM) in chondrogenic medium (CM) for 1 week. Scale bar, 100 μm. (G) RT-qPCR analyses of SOX9, COL2A1, ACAN, RUNX2, COL1A1, and COL10A1 in SMSCs treated with nocodazole (N) and docetaxel (D) in chondrogenic medium (CM) for 1 week. 2.5 nM of docetaxel had significant effects on chondrogenesis in SMSCs. Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.