Fig. 4: Impaired mitochondrial transfer by deleting Rhot1 in osteocytes leads to TCV regression.

a Confocal images show the decreased transfer of mitochondria from Rhot1^KD-MLO-Y4^Mito-Dendra2 cells to CD31-stained bEnd.3 cells after 24 h of coculture. Scale bars, 10 µm. b Workflow for the flow cytometry analysis of the mitochondrial acquisition efficiency in bEnd.3 cells after coculturing with NC-MLO-Y4^Mito-Dendra2 or Rhot1^KD-MLO-Y4 cells. This figure was created by P.L. and D.L.L. and cartonized by Mr. Zihao Li. c Representative dot-plots and quantitative result of the percentage of bEnd.3 cells acquired with Mito-Dendra2 fluorescence in entire bEnd.3 cells population after coculturing with NC-MLO-Y4^Mito-Dendra2 or Rhot1^KD-MLO-Y4 cells (n = 3 biologically independent samples). d Schematic diagram for the generation of the Dmp1^Cre-Cox8^Dendra2-Rhot1^fl/fl mouse line. e Representative confocal images of Dendra2-labeled mitochondria and CD31-labeled endothelial cells from the femur cortical bone of 6-week-old male Dmp1^Cre-Cox8^Dendra2 control mice and Dmp1^Cre-Cox8^Dendra2-Rhot1^fl/fl mice. Scale bars, 20 µm. f Quantitative assessment of the number of Dendra2-labeled mitochondria in each TCV as shown in e. (Data were quantified from three mice, and four different views per mouse were captured for the quantification). g Strategy diagram of the generation of the Dmp1^Cre-Rhot1^fl/fl mouse line for the specific deletion of the Rhot1 gene in Dmp1-expressing cells by crossing the Loxp-flanked Rhot1 allele with the Dmp1^Cre mouse line. h Representative confocal images of CD31-labeled blood vesselsfrom femur cortical bone of 6-week-old male Rhot1^fl/fl control mice or Dmp1^Cre-Rhot1^fl/fl mice. Scale bars, 200 µm. i Quantitative assessment of the number of TCV branches (white arrowhead) shown in h (Data were quantified from three mice, and upper and lower cortical bone of each mouse were captured for the quantification). j Representative images of high-resolution µCT (1 µm resolution) on 6-week-old male mouse femurs presenting the regressed canal network in Dmp1^Cre-Rhot1^fl/fl mice compared to Rhot1^fl/fl control mice. Data were presented as the means ± SEMs. Significance was calculated using unpaired t test with two-tailed P value c, f, i. Source data are provided as a Source Data file.