Related Experiment Video
Updated: Aug 9, 2025

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation
Published on: March 15, 2018
Robo2 promotes osteoblast differentiation and mineralization through autophagy and is activated by parathyroid
Ning Yuan1, Xiaojuan Wang1, Minghai He1
1Department of Endocrinology, Nanchong Central Hospital, Nanchong, Sichuan 637000, China.
Background:
As a systemic skeletal disorder, osteoporosis can increase fracture risk. This study wants to discuss the mechanism of osteoporosis and find possible molecular therapy. Bone morphogenetic protein 2 (BMP2) was utilized to stimulate MC3T3-E1 to establish a cellular osteoporosis model in vitro.
Methods:
Initially, the viability of BMP2-induced MC3T3-E1 was assessed with a Cell counting kit-8 (CCK-8) assay. By real-time quantitative PCR (RT-qPCR) and western blot, Robo2 expression after roundabout (Robo) silencing or overexpression was estimated. Besides, alkaline phosphatase (ALP) expression, mineralization level and LC3II green fluorescent protein (GFP) expression were evaluated using ALP assay, Alizarin red staining and immunofluorescence staining, separately. Additionally, the expression of proteins related to osteoblast differentiation and autophagy was analyzed by RT-qPCR and western blot. Then, following autophagy inhibitor 3-methyladenine (3-MA) treatment, osteoblast differentiation and mineralization were measured again.
Results:
MC3T3-E1 cells were differentiated into osteoblasts under BMP2 induction and Robo2 expression was greatly ascended. After Robo2 silencing, Robo2 expression was markedly diminished. ALP activity and mineralization level in BMP2-induced MC3T3-E1 cells were declined after depleting Robo2. Robo2 expression was conspicuously enhanced after overexpressing Robo2. Robo2 overexpression promoted the differentiation and mineralization of BMP2-induced MC3T3-E1 cells. Rescue experiments revealed that Robo2 silence and its overexpression could regulate the autophagy of BMP2-stimulated MC3T3-E1 cells. After 3-MA treatment, the increased ALP activity and mineralization level of BMP2-induced MC3T3-E1 cells with Robo2 upregulation were reduced. Furthermore, parathyroid hormone 1-34 (PTH1-34) treatment enhanced the expression of ALP, Robo2, LC3II and Beclin-1 and reduced the levels of LC3I and p62 of MC3T3-E1 cells concentration-dependently.
Conclusion:
Collectively, Robo2, which was activated by PTH1-34, promoted osteoblast differentiation and mineralization through autophagy.
More Related Videos
14:55Analysis of Minerals Produced by hFOB 1.19 and Saos-2 Cells Using Transmission Electron Microscopy with Energy Dispersive X-ray Microanalysis
Published on: June 24, 2018
11:11Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow
Published on: May 19, 2019
Related Concept Videos
Hormones and Bone Tissue
Hormones That Influence Osteoblasts and/or Maintain the Matrix
Several hormones are necessary for controlling bone growth and maintaining the bone matrix. The pituitary gland secretes growth hormone (GH), which, as its name implies, controls bone growth. This happens in several ways: first, it triggers chondrocyte...
Bone Remodeling
Osteoclasts in Bone Remodeling
The Parathyroid Glands
Oxyphil cells, whose functions remain elusive, emerge during late puberty, adding a layer of complexity to the parathyroid gland's intricacies. In contrast, principal parathyroid cells undertake a vital role by...
mTOR Signaling and Cancer Progression
The mTOR pathway or the...
Skeleton and Calcium Homeostasis