Figure 5
Figure 5. Wnt5a is involved in osteoblast differentiation in ATL cells. (A) Real-time PCR analysis of Wnt5a expression from cDNA derived from ATL cell lines. Samples were normalized to GAPDH expression and the fold change was calculated by comparing values with R.PBMCs’ normalized gene expression. Jurkat expression served as a negative control. (B) C2C12 and doxycycline-treated MT1-DKK1 or MT1-pTRIPZ cells were cultured in 0.4 µM Transwell plates with or without 100 ng/mL rBMP-2. After 5 days, C2C12 cells were lysed and used in alkaline phosphatase assays. ALP activity was measured as the amount of pNP generated in µmol per volume of the sample per reaction time and normalized to protein concentrations. Results are representative of 2 independent experiments. (C) MT1 cells stably expressing DKK-1 were induced with or without 2 µg/mL doxycycline (Dox) for 72 hours. Real-time PCR was performed on cDNA for DKK-1 expression and normalized to GAPDH expression. Results are representative of 2 independent inductions. (D) C2C12 cells were cocultured with MT1-pTRIPZ cells on 0.4 µM Transwell plates. Three hours after culturing, 50 ng/mL rWnt3a was added and cells were grown at subconfluence for an additional 48 hours. RNA was extracted from C2C12 cells and used for real-time PCR analysis. Results are representative of 2 independent experiments. Samples were normalized to GAPDH expression and the fold change was calculated by comparing values with C2C12 cells without rWnt3a-normalized gene expression. (E) Model demonstrating the C2C12 Transwell system used in the study. MT1 cells were seeded in a Transwell insert and C2C12 cells were seeded at subconfluency on the bottom of the plate. Secreted Wnt5a was blocked by the addition of anti-Wnt5a antibody. (F) C2C12 cells were cultured in a Transwell plate with and without MT1-TRIPZ cells and with or without Wnt5a antibody. Three hours after the addition of MT1-pTRIPZ cells and Wnt5a antibody, 30 ng/mL of rRANKL or control was added. Cells were cultured for a further 48 hours, after which RNA was extracted and used for real-time PCR. Results are representative of 2 independent experiments. Samples were normalized to GAPDH expression and the fold change was calculated to control normalized gene expression.

Wnt5a is involved in osteoblast differentiation in ATL cells. (A) Real-time PCR analysis of Wnt5a expression from cDNA derived from ATL cell lines. Samples were normalized to GAPDH expression and the fold change was calculated by comparing values with R.PBMCs’ normalized gene expression. Jurkat expression served as a negative control. (B) C2C12 and doxycycline-treated MT1-DKK1 or MT1-pTRIPZ cells were cultured in 0.4 µM Transwell plates with or without 100 ng/mL rBMP-2. After 5 days, C2C12 cells were lysed and used in alkaline phosphatase assays. ALP activity was measured as the amount of pNP generated in µmol per volume of the sample per reaction time and normalized to protein concentrations. Results are representative of 2 independent experiments. (C) MT1 cells stably expressing DKK-1 were induced with or without 2 µg/mL doxycycline (Dox) for 72 hours. Real-time PCR was performed on cDNA for DKK-1 expression and normalized to GAPDH expression. Results are representative of 2 independent inductions. (D) C2C12 cells were cocultured with MT1-pTRIPZ cells on 0.4 µM Transwell plates. Three hours after culturing, 50 ng/mL rWnt3a was added and cells were grown at subconfluence for an additional 48 hours. RNA was extracted from C2C12 cells and used for real-time PCR analysis. Results are representative of 2 independent experiments. Samples were normalized to GAPDH expression and the fold change was calculated by comparing values with C2C12 cells without rWnt3a-normalized gene expression. (E) Model demonstrating the C2C12 Transwell system used in the study. MT1 cells were seeded in a Transwell insert and C2C12 cells were seeded at subconfluency on the bottom of the plate. Secreted Wnt5a was blocked by the addition of anti-Wnt5a antibody. (F) C2C12 cells were cultured in a Transwell plate with and without MT1-TRIPZ cells and with or without Wnt5a antibody. Three hours after the addition of MT1-pTRIPZ cells and Wnt5a antibody, 30 ng/mL of rRANKL or control was added. Cells were cultured for a further 48 hours, after which RNA was extracted and used for real-time PCR. Results are representative of 2 independent experiments. Samples were normalized to GAPDH expression and the fold change was calculated to control normalized gene expression.

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