Fig. 3

Elevated ROS in PDE offspring rats originated in the uterus and was of the potential to enhance osteoclast function. A, C Representative images of ROS production staining with DAPI in decalcified bone sections of fetal rats and adult offspring rats. B, D Quantification analyses of ROS mean optical density. E Representative images of ROS production staining in osteoclasts treated with different concentrations of DEX. F Quantification analyses of ROS mean optical density. G Representative images of TRAP and F-actin staining of osteoclasts treated with different concentrations of ROS. H Quantification analyses of TRAP⁺ osteoclastic cells number per well. I Quantification analyses of F-actin belt size per osteoclast. J-O NFATc1, c-Fos, Acp5, CtsK, Oscar, and DC-stamp mRNA expression in osteoclasts treated with different concentrations of ROS. P Representative images of ROS production and TRAP staining of osteoclasts in the 500 nM DEX group with or without TEM. Q Quantification analyses of ROS mean optical density. R Quantification analyses of TRAP⁺ osteoclastic cells number per well. S Experimental protocol for alternate treatment of osteoclasts with DEX and ROS. T Representative images of TRAP staining of osteoclasts treated with Fig. 4M protocol. U Quantification analyses of TRAP⁺ osteoclastic cells number per well. Mean ± S.E.M., n = 3 per group for ROS production, TRAP and F-actin staining, n = 6 per group for mRNA expression. ^*P < 0.05, ^**P < 0.01 vs. Control in vivo or 0 nM DEX in vitro. ^##P < 0.01 vs. 500 nM DEX. ROS: reactive oxygen species; PDE: prenatal dexamethasone exposure; DEX: dexamethasone; TRAP: tartrate-resistant acid phosphatase; NFATc1: nuclear factor of active T cells 1; c-Fos: protooncogene c-Fos; Acp5: acid phosphatase 5; CtsK: cathepsin K; Oscar: osteoclast-associated receptor; DC-stamp: dendritic cell-specific transmembrane protein; DEX: dexamethasone; TEM: tempol; GAPDH: glyceraldehyde-3-phosphate dehydrogenase