Figure 5.

Lentiviral overexpression of BMP4 in two USSC and two CBSC cell lines. With regard to the staining, only one representative cell line per cell type is shown. (a) Quantitative RT-PCR analysis of gene expression (in undifferentiated cells) after overexpression in relation to the control cells (Mock). RPL13A was used as housekeeping gene. (b): Representative illustration of the Oil Red O staining after adipogenic differentiation (21 d) of BMP4-transfected and control-cells. +: induced cells; −: non-induced cells. Lipid vacuoles are stained red. Scale bar: 100 μm. (c) ImageJ-based quantification of Oil Red O staining. *P = 0.01 to 0.05, significant (unpaired t-test). (d) Osteogenic differentiation experiments in overexpressed and Mock cells. After 14 days of differentiation, Von Kossa and Alizarin Red S staining were performed in the induced (+) and non-induced (−) cells. Mineralized areas are stained in brown/black or red, respectively. Scale bar: 200 μm. (e) Subsequent quantification of the bound Alizarin Red S-dye. **P = 0.001 to 0.005, very significant; ***P < 0,001, extremely significant (unpaired t-test). (f) Safranin O staining of transfected and control cells after chondrogenic differentiation in pellet culture for 21 days. Proteoglycans are stained purple/red. Scale bar: 200 μm. BMP4: bone morphogenetic protein 4, LRP5: low-density lipoprotein receptor-related protein 5, RUNX2: runt-related transcription factor 2, and RPL13A: ribosomal protein L13A.