Additional file 1 of RhoA promotes osteoclastogenesis and regulates bone remodeling through mTOR-NFATc1 signaling
Creators: name: Wang, Jirong
name: Xu, Chengyun
name: Zhang, Jing
name: Bao, Yizhong
name: Tang, Ying
name: Lv, Xiaoling
name: Ma, Bo
name: Wu, Ximei
name: Mao, Genxiang
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Environmental Sciences not elsewhere classified
subject: Ecology
subject: Biological Sciences not elsewhere classified
subject: Mathematical Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Hematology
subject: Plant Biology

description: Additional file 1. Figure S1. Knock down of RhoA inhibits osteoclastogenesis in RAW264.7 cells. (a) The effect of RANKL and M-CSF stimulation on RhoA activity in pre-osteoclasts. (b) The qRT-PCR analysis following scrambling small hairpin RNA (shScr.) and RhoA shRNA (shRhoA) knockdown, n = 3. (c) Representative TRAP staining images of shRhoA infected RAW264.7 cells treated by RANKL for 4 days, n = 6, Scale bars, 200 μm. (d) The expression levels of the osteoclast-specific gene in shRhoA infected RAW264.7 cells for indicated times carried out with qRT-PCR, n=3. Mean± s.d., +,*P &lt; 0.05, ++,**P &lt; 0.01, Student’s t-test or ANOVA analysis. Figure S2. Micro-CT measurement of cortical bone. (a) The western blotting of RhoA in BMMs from LCre;RhoAf/f and RCre;RhoAf/f mice. (b) The qRT-PCR of RhoA in BMMs from LCre;RhoAf/f and RCre;RhoAf/f mice. (c, e) 3D Micro-CT reconstructions of the tibial midshaft region. (d, f) Micro-CT measurement of cortical bone structural parameters from the midshaft region. n=6, Tt.Ar, total cross-sectional area; Ct.Ar, cortical bone area; Ct.Th, cortical thickness. Mean± s.d., **P &lt; 0.01, Student’s t-test. Figure S3. Loss of RhoA in osteoclast inhibited osteoclastgenesis. (a) Representative TRAP staining images of BMMs from RhoAf/f and RCre;RhoAf/f mice cultured with M-CSF and RANKL. The osteoclast numbers were counted (right). n=6, Scale bars, 100 µm. (b) Expression of osteoclast-specific genes in RhoAf/f and RCre;RhoAf/f mice osteoclasts, n=6. (c) Representative Phalloidin staining images of BMMs, and the quantification of F-actin ring per well and nuclei per osteoclast. n=6, Scale bars, 50 µm. (d) SEM analysis and quantification of bone resorption area of bone slides, n=6, Scale bars, 20 µm. Mean±s.d., *P&lt; 0.05, Student’s t-test or ANOVA analysis. Figure S4. RhoA gain-of-function in the early stage of osteoclast differentiation enhanced osteoclast activity and decreases bone mass. (a) Representative Micro-CT images of 3-month-old caRhoA and RCre;caRhoA mice tibias. (b) The parameters of tibias growth plate trabecular bone, n=6. (c) Representative H&amp;E and TRAP staining images of mice femurs. Scale bars, 200 µm (H&amp;E) and 20 µm (TRAP). (d) The parameters of femur osteoclasts, n=6. (e) Representative TRAP staining images of BMMs from caRhoA and RCre;caRhoA mice. Scale bars, 200 µm. (f) SEM analysis and quantification of bone resorption area of bone slides, n=6, Scale bars, 20 µm. (g) Representative Phalloidin staining images and quantification of F-actin ring per well and nuclei per osteoclast, n=3, Scale bars, 500 µm. Mean±s.d., *P&lt; 0.05, Student’s t-test.