Figure 7.

EMTT stimulation enhanced mineralization and upregulated mineralization-related genes in human osteoblasts (hOBs). (a) Alizarin Red S staining. (b) Von Kossa staining. Two representative wells are illustrated. (c,d) The images were quantitated using ImageJ software. (e) The Alizarin Red dye was eluted using cetylpyridinium chloride, and the absorbance was measured at 405 nm. Results are presented relative to the control (normalized to 1). (f–h) Data analysis was performed by using the 2^−ΔΔCT method. Gene expression was normalized to GAPDH and compared by setting control cultures to 1 as a reference value. (f–h) Cells were stimulated with EMTT according to our stimulation protocol (Figure 1b), followed by RNA extraction and PCR. Data are expressed as the average ± SD of three to six independent experiments (n = 3–6). ns: non-significant. * p < 0.05, ** p < 0.01, and *** p < 0.001, two-tailed Student’s t-test.