Journal: Nature Communications

Article Title: Osteoclasts protect bone blood vessels against senescence through the angiogenin/plexin-B2 axis

doi: 10.1038/s41467-021-22131-1

Figure Lengend Snippet: a–d Three-week-old C57BL/6J mice were treated with MPS at 10 mg/m 2 /day or vehicle by daily intraperitoneal injection for 2 weeks. Double immunofluorescence staining of femoral sections using antibodies against PLXNB2 (green) and Emcn (red) or Osx (red) ( a ). Simultaneous fluorescence immunostaining of Emcn (red) and FISH analysis of 47S rRNA transcription (green) in bone tissue sections were shown in ( b ). DAPI stains nuclei blue. Boxed areas are shown at a higher magnification in corresponding panels to the right. Quantitative analysis of percentage of Emcn + blood vessels in ( c ) and non-blood vessel cells ( d ) that show positive signal of 47S rRNA transcription in primary spongiosa. Blood vessels with more than 20 green dots are considered as positive. Non-blood vessel cell with more than five green dots in nucleus are considered positive. e Immunoblot of PLXNB2 in HUVECs transfected with two individual PLXNB2 siRNAs (siPLXNB2-1, -2) or scrambled control siRNA (siCTRL). f–i HUVECs were transfected with siCTRL or siPLXNB2 for 36 h and then stimulated with 200 ng/mL rhANG or vehicle for another 12 h. Cells were fixed and immunofluorescence staining was performed using antibody against ANG (red) in ( f ). Arrow heads indicate nuclear translocation of ANG. DAPI stains nuclei blue. Immunoblot was performed using antibodies against p-AKT, AKT, p-ERK, or ERK in ( g ). Click-iT RNA-imaging assays were performed, and confocal images of the cells pulsed with EU for 2 and 6 h were obtained (green: Alexa 488-EU) in ( h ). Cells were subjected to SA-βGal staining with representative images in ( i ). j HUVECs were treated transfected with siCTRL or siPLXNB2 for 48 h. Immunoblot was performed using individual antibodies against LaminB1, Ki67, p21, and GAPDH. k , l HUVEC was seeded on Matrigel with control IgG, 26-2F or mAb17. Images of tubule formation is shown in ( k ). Quantitative analysis of cumulative tubule length is shown in ( l ). ** p < 0.01 as determined by one-way ANOVA with post hoc Tukey test. GP growth plate. n = 4-6 mice, Data are represented as mean ± s.e.m. ** p < 0.01, ns not significant as determined by two-tailed Student’s t -tests.

Article Snippet: For ANG administration, 3-week-old BALB/c mice were treated with rhANG (1 µg/day, R&D systems, 265-AN) as previously described .

Techniques: Injection, Double Immunofluorescence Staining, Fluorescence, Immunostaining, Western Blot, Transfection, Immunofluorescence, Staining, Translocation Assay, Imaging, Two Tailed Test

Journal: Nature Communications

Article Title: Osteoclasts protect bone blood vessels against senescence through the angiogenin/plexin-B2 axis

doi: 10.1038/s41467-021-22131-1

Figure Lengend Snippet: Three-week-old BALB/c mice were treated with vehicle, MPS alone at 10 mg/m 2 /day or MPS plus rhANG (1 µg/day) by daily intraperitoneal injection for 4 weeks. Representative images of SA-βGal staining (white) and immunofluorescence staining of Endomucin (Emcn, red) in primary spongiosa of femoral bone in ( a ). Percentage of SA-βGal-expressing vessels were quantified in ( b ). Double immunofluorescence staining of Emcn (red) and CD31 (green) in metaphysis of femoral bone in ( c ). DAPI-stained nuclei blue. Relative yellow fluorescence intensity in primary spongiosa was measured in ( d ). Immunofluorescence staining of femoral metaphysis sections were performed using antibody against Osx (red) in ( e ). DAPI stains nuclei blue. Numbers of Osx + cells per mm 2 tissue area (N. Osx + cells/Ar) were quantified in ( f ). Representative μCT images of distal femur in mice were shown in ( g , longitudinal sections) and ( h , cross sections). Quantitative analyses of trabecular bone volume fraction (BV/TV) ( i ), trabecular thickness (Tb. Th) ( j ), trabecular number (Tb. N) ( k ), and trabecular separation (Tb.Sp) ( l ). Trichrome staining of the metaphyseal trabecular bone at distal femora in ( m ). Osteoid stains red and mineralized bone stains green. Osteoid surface per bone surface (OS/BS) was measured in ( n ). Representative images of calcein double labeling ( o ) and quantification of bone formation rate per bone surface (BFR/BS) ( p ) of the metaphyseal trabecular bone at distal femora. GP growth plate. n = 6-10 mice. Data are represented as mean ± s.e.m. ** p < 0.01 as determined by one-way ANOVA with post hoc Tukey test.

Article Snippet: For ANG administration, 3-week-old BALB/c mice were treated with rhANG (1 µg/day, R&D systems, 265-AN) as previously described .

Techniques: Injection, Staining, Immunofluorescence, Expressing, Double Immunofluorescence Staining, Fluorescence, Labeling