Alkaline Phosphatase Antibody - #DF6225

Figure 7. The dynamic HA-ADA hydrogel enhances the osteogenic differentiation. D) Representative images and E) quantified intensity (n = 20) of ALP (green) immunostaining of the hMSCs in the hydrogels.

Figure 8 Validation of ECM1/ENO1/MAPK signaling axis in patients with bmCRPC. A) Representative images (left) of PCa PDOs treated with Veh (PBS), ECM1 (200 ng mL−1), or ECM1 (200 ng mL−1) combined with either DMSO or PhAH (1 µM) in the presence of ENZ (10 µM). B) Assessment of PDOs growth by changes in organoid diameter (right; Scale bar, 50 µm). C) IHC staining of ALP, ECM1, ENO1, and p-ERK1/2 in ENZ-untreated (n = 12) or ENZ-treated (n = 22) bmCRPC patients tissue (Scale bars, 250 µm and 50 µm). D-G) Staining index quantification of ALP, ECM1, p-ERK1/2 expression and relative ENO1 membrane/cytoplasm cell number as indicated in C. H) Percentage of specimens showing ENZ-untreated or treated in relation to the expression levels of ALP, ECM1, p-ERK1/2 and the membrane or cytoplasm level of ENO1. I-K) Spearman correlation analysis between the expression levels of ALP, relative ENO1 membrane/cytoplasm cell number, and p-ERK1/2 with ECM1 expression. L) Representative mIHC staining images of ALP (red, osteoblast marker), EPCAM (yellow, PCa marker), and ENO1 (green) in human bmCRPC tissues (Scale bars, 100 µm and 25 µm). M) Quantification of ALP, EPCAM, and relative ENO1 membrane/cytoplasm intensity per cell in L). N) WB analysis of MEK, p-MEK, ERK1/2, and p-ERK1/2 expression in prostate cancer cells extracted from human bmCRPC tissue with or without ECM1 treatment (200 ng mL−1). GAPDH was used as the loading control. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 8 Validation of ECM1/ENO1/MAPK signaling axis in patients with bmCRPC. A) Representative images (left) of PCa PDOs treated with Veh (PBS), ECM1 (200 ng mL−1), or ECM1 (200 ng mL−1) combined with either DMSO or PhAH (1 µM) in the presence of ENZ (10 µM). B) Assessment of PDOs growth by changes in organoid diameter (right; Scale bar, 50 µm). C) IHC staining of ALP, ECM1, ENO1, and p-ERK1/2 in ENZ-untreated (n = 12) or ENZ-treated (n = 22) bmCRPC patients tissue (Scale bars, 250 µm and 50 µm). D-G) Staining index quantification of ALP, ECM1, p-ERK1/2 expression and relative ENO1 membrane/cytoplasm cell number as indicated in C. H) Percentage of specimens showing ENZ-untreated or treated in relation to the expression levels of ALP, ECM1, p-ERK1/2 and the membrane or cytoplasm level of ENO1. I-K) Spearman correlation analysis between the expression levels of ALP, relative ENO1 membrane/cytoplasm cell number, and p-ERK1/2 with ECM1 expression. L) Representative mIHC staining images of ALP (red, osteoblast marker), EPCAM (yellow, PCa marker), and ENO1 (green) in human bmCRPC tissues (Scale bars, 100 µm and 25 µm). M) Quantification of ALP, EPCAM, and relative ENO1 membrane/cytoplasm intensity per cell in L). N) WB analysis of MEK, p-MEK, ERK1/2, and p-ERK1/2 expression in prostate cancer cells extracted from human bmCRPC tissue with or without ECM1 treatment (200 ng mL−1). GAPDH was used as the loading control. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Figure 1. POL stimulated BMSCs proliferation and osteogenic differentiation. (A) POL chemical structure; (B) BMSCs surface markers were detected by flow cytometry; (C) The result of CCK-8 assay (n = 5); (D-E) The result of CFU assay (n = 5); (F) Images of ALP staining (scale bar = 250 μm); (G) Quantification of ALP staining (n = 5); (H) Images of ARS staining (scale bar = 250 μm); (I) Quantification of ARS staining (n = 5); (J-K) The protein expression levels of OCN, RUNX2, ALP, and COL1A1 were evaluated by western blot (n = 3); (L-M) Immunofluorescence staining of OCN and ALP (scale bar = 200 μm). Data were presented as mean ± SEM. Compared with control group: * P < 0.05, **P < 0.01, ***P < 0.001. Compared with 1 μM group: # P < 0.05, ##P < 0.01, ###P < 0.001.

Figure 3 circRNA422 interfering inhibited osteogenic differentiation of BMSCs (A) ALP staining to evaluate the effect of circRNA422− on the ALP activity of BMSCs on osteogenic days 3 and 7. (B) Protein levels of OCN to evaluate the effect of circRNA422− on mineralization of BMSCs on osteogenic days 14 and 21. (C) Quantitative real-time PCR analysis showed that the osteogenic mRNA expressions levels of SP7, LRP5, ALP, OCN, BSP, and OPN were downregulated by circRNA422− compared with Con313 on osteogenic days 3 and 7 (n = 3, ∗p < 0.05). (D) WB analysis showed that the osteogenic protein expressions levels of ALP, LRP5, and SP7 were reduced by circRNA422− compared with Con313 on osteogenic days 3 and 7 (n = 3, ∗p < 0.05).