Journal: Scientific reports

Article Title: Hem1 is essential for ruffled border formation in osteoclasts and efficient bone resorption.

doi: 10.1038/s41598-024-58110-x

Figure Lengend Snippet: Figure 1. Hem1-deficiency leads to osteopetrosis-like phenotype. (A) Hem1-/- mice display signs of growth retardation and reduced femoral length. (B) Representative µCT cross sections of distal femora of WT and Hem1-/- animals. (C) Statistical analysis of trabecular bone volume to tissue volume (BV/TV,) trabecular thickness (Tb.Th.), trabecular number (Tb.N.) and trabecular separation (Tb.Sp.) of trabecular bone from 8–12 weeks old WT and Hem1-/- male mice. **** P < 0.0001 (BV/TV, Tb.Th.), ** = P 0.0062 (Tb.N.), *** P = 0.0005 (Tb.Sp.), two-tailed Mann–Whitney U test. (D) Representative H&E staining of distal femora from WT and Hem1-/- mice, red box represents magnified area. (E) Left: Representative TRAP staining of distal femora from WT and Hem1-/- mice, red box represents magnified area. Right: Quantification of TRAP-positive osteoclasts within trabecular region from distal femora of WT and Hem1-/- mice. * P = 0.0317, two-tailed Mann– Whitney U test. All data presented as mean ± SEM.

Article Snippet: Specific antibodies against the following proteins were detected by immunoblotting: cathepsin K (abcam, ab 19,027, 1:1000), integrin alpha-v (abcam, ab179475,1:1000), integrin beta-3 (Cell Signaling #4702,1:1000), DC-STAMP (sc-98769, 1:200), NFATc1 (sc-7294; both from Santa Cruz), Hem1 (Novus Bio, NBP2-13,643, 1:1000), WASP (Santa Cruz, sc-13139, 1:1000) and WAVE2 (Cell Signaling, #3659, 1:1000).

Techniques: Two Tailed Test, MANN-WHITNEY, Staining

Journal: Scientific reports

Article Title: Hem1 is essential for ruffled border formation in osteoclasts and efficient bone resorption.

doi: 10.1038/s41598-024-58110-x

Figure Lengend Snippet: Figure 2. Hem1-deficiency promotes osteoclastogenesis but inhibits resorptive capacity. (A) In-vitro osteoclast differentiation assay of WT and Hem1-/- osteoclasts visualized by tartrate resistant acid phosphatase (TRAP) staining. Representative images for both genotypes are shown, scale bar 200 µm. (B) Quantification of TRAP-assay of WT and Hem1-/- shown in (A). * P = 0.0234, ** P = 0.0078, two-tailed Wilcoxon matched-pairs signed rank test. (C) Representative images of WT and Hem1-/- osteoclasts cultured on calcium phosphate (CaPO4 2-; CaP plates) before lysis. Osteoclasts indicated with blue arrows, resorption pits with red asterisk. Scale bar 100 µm. (D) Representative images of resorption pit formation of WT and Hem1-/- osteoclasts using calcium phosphate on CaP-coated substrate. Scale bar 100 µm. (E) Quantification of (D): total resorbed area/well, number of resorption pits/well, mean pit size/well and percentage of resorbed area/well. ** P = 0.0079, * P = 0.0159, two-tailed Mann–Whitney U test. All data presented as mean ± SEM. (F) Immunoblotting of osteoclast markers from WT and Hem1-/- osteoclast lysates, representative images from n = 4 independent experiments. (G) Immunoblotting of Hem1, WAVE2 and WASP from WT and Hem1-/- osteoclast lysates, representative images from n = 5 independent experiments.

Article Snippet: Specific antibodies against the following proteins were detected by immunoblotting: cathepsin K (abcam, ab 19,027, 1:1000), integrin alpha-v (abcam, ab179475,1:1000), integrin beta-3 (Cell Signaling #4702,1:1000), DC-STAMP (sc-98769, 1:200), NFATc1 (sc-7294; both from Santa Cruz), Hem1 (Novus Bio, NBP2-13,643, 1:1000), WASP (Santa Cruz, sc-13139, 1:1000) and WAVE2 (Cell Signaling, #3659, 1:1000).

Techniques: In Vitro, Osteoclast differentiation Assay, Staining, TRAP Assay, Two Tailed Test, Cell Culture, Lysis, MANN-WHITNEY, Western Blot

Journal: Scientific reports

Article Title: Hem1 is essential for ruffled border formation in osteoclasts and efficient bone resorption.

doi: 10.1038/s41598-024-58110-x

Figure Lengend Snippet: Figure 3. Hem1 is essential for ruffled border formation and actin ring organization. (A) Transmission electron microscopy cross-section of WT and Hem1-/- osteoclasts cultured on bovine bone slices. Scale bar 5 µm. Yellow arrows on higher magnification images indicate vesicles close to the basolateral membrane. (B) Immunofluorescent visualization of F-actin (green) and nuclei (blue) in osteoclasts cultured on plastic (upper panel) or CaPO4 2- (lower panel). Scale bar 100 µm (upper panel) 50 µm (lower panel), respectively. Representative images of n = 5 independent experiments. (C) Confocal z-stack images showing actin rings in osteoclasts cultured on plastic (upper panel) or CaPO4 2- (lower panel) visualized by F-actin staining. Scale bar 50 µm. Representative images of n = 3 (upper panel) and n = 4 (lower panel) independent experiments. (D) Percentage of disturbed actin rings in OC cultured on CaPO4 2- and analysis of F-actin intensity throughout the individual actin rings. Representative analysis of n = 4 independent experiments. (E) Left: visualization of acidic vesicles (green) in mature WT and Hem1-/- osteoclasts with LysoSensor™ Green DND-189. Scale bar 50 µm. Right: quantification of fluorescent intensity n = 5. ** P = 0.0079, Wilcoxon matched-pairs signed rank test. All data presented as mean ± SEM.

Article Snippet: Specific antibodies against the following proteins were detected by immunoblotting: cathepsin K (abcam, ab 19,027, 1:1000), integrin alpha-v (abcam, ab179475,1:1000), integrin beta-3 (Cell Signaling #4702,1:1000), DC-STAMP (sc-98769, 1:200), NFATc1 (sc-7294; both from Santa Cruz), Hem1 (Novus Bio, NBP2-13,643, 1:1000), WASP (Santa Cruz, sc-13139, 1:1000) and WAVE2 (Cell Signaling, #3659, 1:1000).

Techniques: Transmission Assay, Electron Microscopy, Cell Culture, Membrane, Staining

Journal: Scientific reports

Article Title: Hem1 is essential for ruffled border formation in osteoclasts and efficient bone resorption.

doi: 10.1038/s41598-024-58110-x

Figure Lengend Snippet: Figure 4. Distribution of Rab7 in WT and Hem1-/- osteoclasts. (A) Representative immunofluorescent staining of Rab7 in WT and Hem1-/- osteoclasts cultured on plastic. Scale bar 100 µm. (B) Immunogold labeling of Rab7 on cross-sections of WT and Hem1-/- osteoclasts cultured on bovine bone slices. Black arrows indicate Rab7 antibodies coupled to gold beads. Scale bar 5 µm.

Article Snippet: Specific antibodies against the following proteins were detected by immunoblotting: cathepsin K (abcam, ab 19,027, 1:1000), integrin alpha-v (abcam, ab179475,1:1000), integrin beta-3 (Cell Signaling #4702,1:1000), DC-STAMP (sc-98769, 1:200), NFATc1 (sc-7294; both from Santa Cruz), Hem1 (Novus Bio, NBP2-13,643, 1:1000), WASP (Santa Cruz, sc-13139, 1:1000) and WAVE2 (Cell Signaling, #3659, 1:1000).

Techniques: Staining, Cell Culture, Labeling

Journal: Scientific reports

Article Title: Hem1 is essential for ruffled border formation in osteoclasts and efficient bone resorption.

doi: 10.1038/s41598-024-58110-x

Figure Lengend Snippet: Figure 5. Hem1 deletion in osteoclasts is accountable for osteoclast dysfunction leading to high bone mass. (A) Osteoclast-specific deletion of Hem1 does not affect overall mouse phenotype. (B) Immunoblotting of Hem1 from Hem1fl/fl, Hem1d/dCtskcre/+, Ctskcre/+ osteoclast lysates, representative images from n = 5 independent experiments. (C) Representative µCT cross sections of distal femora of Hem1fl/fl, Hem1d/dCtskcre/+, Ctskcre/+ animals. Statistical analysis of trabecular bone volume to tissue volume (BV/TV,) trabecular thickness (Tb.Th.), trabecular number (Tb.N.) and trabecular separation (Tb.Sp.) of trabecular bone from 10–12 weeks old Hem1fl/fl, Hem1d/dCtskcre/+, Ctskcre/+ male mice. ** = P 0.0022 (BV/TV), *** P = 0.0004 (BV/TV), **** P < 0.0001 (Tb.N, Tb.Sp.), two-tailed Mann– Whitney U test. (D) Representative H&E staining of distal femora from Hem1fl/fl, Hem1d/dCtskcre/+, Ctskcre/+ mice, red box represents magnified area. (E) Left: representative TRAP staining of distal femora from Hem1fl/fl, Hem1d/dCtskcre/+, Ctskcre/+ mice, red box represents magnified area. Right: Quantification of TRAP-positive osteoclasts within trabecular region from distal femora of Hem1fl/

Article Snippet: Specific antibodies against the following proteins were detected by immunoblotting: cathepsin K (abcam, ab 19,027, 1:1000), integrin alpha-v (abcam, ab179475,1:1000), integrin beta-3 (Cell Signaling #4702,1:1000), DC-STAMP (sc-98769, 1:200), NFATc1 (sc-7294; both from Santa Cruz), Hem1 (Novus Bio, NBP2-13,643, 1:1000), WASP (Santa Cruz, sc-13139, 1:1000) and WAVE2 (Cell Signaling, #3659, 1:1000).

Techniques: Western Blot, Two Tailed Test, MANN-WHITNEY, Staining