Figure 4. Hydrogen sulfide alleviated the inhibitory effect of osteoblast differentiation, proliferation, and survival by Dex. (A) Runx2 expression and localization (red arrow) in the distal end of intact femurs of each experimental group through immunohistochemistry. (Scale bars 50 μm). (B–C) As shown by ALP staining (day 7) (x20) and ALP activity detection (day 7), GYY4137 attenuated the effect of Dex on the inhibition of osteogenic differentiation in primary osteoblasts. (D) Western blot analysis of Runx2 and Osterix expression in rat primary osteoblasts pretreated with Dex and/or GYY4137. n=3, *p<0.05, **p<0.01. (E) The proliferation of rat primary osteoblasts cells was measured by CCK8 assay after cells were treated with Dex and/or GYY4137 from day 1 to day 3. n=3, *p<0.05, **p<0.01. (F) Representative photomicrographs (x200) of EdU staining (top panels) and corresponding total cell photomicrographs (middle panels). Blue: Hochest labeling of cell nuclei; red: EdU labeling of nuclei of proliferative cells. (G) Quantitative data showing the percentages of EdU-positive cells in different treatment groups (number of red versus number of blue nuclei). n=3, *p<0.05, **p<0.01.