hem1  (Santa Cruz Biotechnology)

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

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: 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: 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 (CaPO 4 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: 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 CaPO 4 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 CaPO 4 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 CaPO 4 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: 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: 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 Hem1 fl/fl , Hem1 d/d Ctsk cre/+ , Ctsk cre/+ osteoclast lysates, representative images from n = 5 independent experiments. ( C ) Representative µCT cross sections of distal femora of Hem1 fl/fl , Hem1 d/d Ctsk cre/+ , Ctsk cre/+ 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 Hem1 fl/fl , Hem1 d/d Ctsk cre/+ , Ctsk cre/+ 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 Hem1 fl/fl , Hem1 d/d Ctsk cre/+ , Ctsk cre/+ mice, red box represents magnified area. ( E ) Left: representative TRAP staining of distal femora from Hem1 fl/fl , Hem1 d/d Ctsk cre/+ , Ctsk cre/+ mice, red box represents magnified area. Right: Quantification of TRAP-positive osteoclasts within trabecular region from distal femora of Hem1 fl/fl , Hem1 d/d Ctsk cre/+ , Ctsk cre/+ mice. * P = 0.02, ** P = 0.0013, two-tailed Mann–Whitney U test. All data presented as mean ± SEM. ( F ) Quantification of in-vitro osteoclast differentiation TRAP-assay of Hem1 fl/fl , Hem1 d/d Ctsk cre/+ , Ctsk cre/+ osteoclasts. ( G ) Quantification of resorption pit formation of Hem1 fl/fl , Hem1 d/d Ctsk cre/+ , Ctsk cre/+ osteoclasts cultured on CaP-coated substrate. * P = 0.0286, 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: Western Blot, Two Tailed Test, MANN-WHITNEY, Staining, In Vitro, TRAP Assay, Cell Culture

Journal: Cells

Article Title: WAVE2 Regulates Actin-Dependent Processes Induced by the B Cell Antigen Receptor and Integrins

doi: 10.3390/cells12232704

Figure Lengend Snippet: WAVE2 KD does not reduce the expression of Hem1 protein and does not affect BCR signaling in response to immobilized anti-Ig. ( A ) A20 cells were transfected with control siRNA or WAVE2 siRNA, cultured for 48 h, and then analyzed via immunoblotting with Abs to WAVE2, Hem1, or actin (loading control). Normalized levels of WAVE2 or Hem1 relative to control siRNA-transfected cells are shown. ( B ) A20 cells that had been transfected with control siRNA or WAVE2 siRNA were allowed to spread on anti-IgG-coated tissue culture wells for the indicated times before analyzing BCR signaling by immunoblotting for the phosphorylation of the CD79a ITAM (pCD79a) or the phosphorylated (activated) forms of ERK (pERK) or Akt (pAkt). The same cell lysates were probed for total CD79a, ERK, or Akt. The dashed red line was overlaid on the images of the blots to visually separate the time courses for the control siRNA and WAVE2 siRNA-transfected cells. For both panels, one of three independent experiments with similar results is shown. Full uncropped blots are shown in .

Article Snippet: The membranes were blocked with 5% milk powder in Tris-buffered saline (10 mM of Tris-HCl, pH 8, and 150 of mM NaCl) and then incubated overnight at 4 °C with rabbit Abs to WAVE2 (Thermo Fisher, Waltham, MA, USA #PA5-60975, 1:200 dilution), phosphorylated CD79a (pCD79a; Cell Signaling Technologies, Danvers, MA, USA #5173, 1:1000), CD79a ([ ]; 1:5000), phospho-ERK (pERK; Cell Signaling Technologies, Danvers, MA, USA #9101, 1:1000), ERK (Cell Signaling Technologies, Danvers, MA, USA #9102; 1:1000), phospho-Akt S473 (pAkt; Cell Signaling Technologies, Danvers, MA, USA #9271; 1:1000), Akt (Cell Signaling Technologies, Danvers, MA, USA #9272; 1:1000), or NCKAP1L/Hem1 (Thermo Fisher, Waltham, MA, USA #PA5-58813; 1:500).

Techniques: Expressing, Transfection, Cell Culture, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Hematopoietic cytoplasmic adaptor protein Hem1 promotes osteoclast fusion and bone resorption in mice

doi: 10.1016/j.jbc.2022.102841

Figure Lengend Snippet: Deletion of Hem1 increases trabecular bone mass in mice. Micro-CT imaging and quantification of femoral bones from male Hem1 knockout mice and wildtype littermates at 5.5 weeks of age. A , representative images of whole femur ( left ) and length measurement ( right ). B , representative images of cortical bone at midshaft (scale bar represents 100 μm). C , cortical thickness and cortical perimeter at midshaft. D , representative images of trabecular bone (scale bar represents 1 mm) and ( E ) bone volume per tissue volume (BV/TV), bone mineral density (BMD), and microarchitecture of trabecular bone. F , histological sections of distal femurs stained with Masson's trichrome. G , standard histomorphometric parameters quantifying trabecular architecture (BV/TV, trabecular number, thickness, and spacing) in histologic sections of 5.5-week-old male mice. Lines and error bars represent mean ± SD; n = 4 to 5 animals/group. p Values were determined with Student’s t test. Hem1, hematopoietic protein 1.

Article Snippet: Also, we used polyclonal antibodies for Hem1 (Novus Biologicals; catalog no.: NBP2-13643, 1/1000 dilution), c-Fos (Santa Cruz Biotechnology; catalog no.: sc-7202, 1/500 dilution), p38 (Cell Signaling; catalog no.: 9212, 1/1000 dilution), IκB (Santa Cruz Biotechnology; catalog no.: sc-847, 1/500 dilution), Akt (Cell Signaling; catalog no.: 9272, 1/1000 dilution), and p-CrkL (Cell Signaling Technology; catalog no.: 3181, 1/1000 dilution) to analyze their protein levels.

Techniques: Micro-CT, Imaging, Knock-Out, Staining

Journal: The Journal of Biological Chemistry

Article Title: Hematopoietic cytoplasmic adaptor protein Hem1 promotes osteoclast fusion and bone resorption in mice

doi: 10.1016/j.jbc.2022.102841

Figure Lengend Snippet: Deletion of Hem1 in mice decreases bone resorption. A , representative photomicrographs of osteoclasts ( left ), number of osteoclasts ( middle ; N.Oc/B.Pm), and nuclei number per osteoclast ( right ; N.Nuclei/Oc) per trabecular bone surface of nondecalcified femur sections stained for TRAP activity ( red arrows ) from 5.5-week-old male Hem1 knockout mice and wildtype littermates (n = 4–5 animals/group) (scale bar represents 400 μm). Dotted red lines indicate multinucleated TRAP-positive osteoclasts. B , ELISA analysis of serum concentration of a collagen degradation product (CTx) in 5.5-week-old male Hem1 knockout mice and wildtype littermates (n = 4–5 animals/group). C , representative photomicrographs of osteoblasts ( left ; black arrows ) and number of osteoblasts ( right ; N.Ob/B.Pm) per trabecular bone surface of nondecalcified femur sections stained for Masson's trichrome from 5.5-week-old male Hem1 knockout mice and wildtype littermates (n = 4–5 animals/group) (scale bar represents 100 μm). D , ELISA analysis of serum concentration of osteocalcin (n = 3–4 animals/group). E – G , photomicrographs show representative colonies ( top ). CFU-F stained for alkaline phosphatase after 10 days, CFU-AD stained with Oil Red O after 7 days, and CFU-OB stained with von Kossa after 25 days to detect mineral ( bottom ) (triplicate cultures) (scale bar represents 1 cm). Lines and error bars represent mean ± SD. p Values were determined with Student’s t test. CFU-AD, CFU adipocyte; CFU-F, CFU fibroblast; CFU-OB, CFU-osteoblast; Hem1, hematopoietic protein 1; TRAP, tartrate-resistant acid phosphatase.

Article Snippet: Also, we used polyclonal antibodies for Hem1 (Novus Biologicals; catalog no.: NBP2-13643, 1/1000 dilution), c-Fos (Santa Cruz Biotechnology; catalog no.: sc-7202, 1/500 dilution), p38 (Cell Signaling; catalog no.: 9212, 1/1000 dilution), IκB (Santa Cruz Biotechnology; catalog no.: sc-847, 1/500 dilution), Akt (Cell Signaling; catalog no.: 9272, 1/1000 dilution), and p-CrkL (Cell Signaling Technology; catalog no.: 3181, 1/1000 dilution) to analyze their protein levels.

Techniques: Staining, Activity Assay, Knock-Out, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Hematopoietic cytoplasmic adaptor protein Hem1 promotes osteoclast fusion and bone resorption in mice

doi: 10.1016/j.jbc.2022.102841

Figure Lengend Snippet: Deletion of Hem1 decreases osteoclast fusion. A and B , bone marrow macrophages were isolated from 6-month-old C57BL/6 wildtype mice and were cultured with M-CSF (30 ng ml −1 , bone marrow macrophages) or with M-CSF and RANKL (30 ng ml −1 ) for 2 days (pOC) or 5 days (mOC). Levels of Hem1 ( A ) mRNA (quantitative PCR [qPCR] assay) and ( B ) protein (Western blot) during osteoclastogenesis. C – H , bone marrow macrophages were isolated from 5.5-week-old male Hem1 knockout mice and wildtype littermates and were cultured with M-CSF (30 ng ml −1 ) and RANKL (30 ng ml −1 ) for ( C ) 24 h, ( D , F , and G) 5 days, ( H ) 3 days, or ( E and H ) indicated periods. C , Hem1 mRNA levels (qPCR assay) during osteoclastogenesis. D , representative pictures ( left ) and number ( right ) of TRAP-positive multinucleated osteoclasts generated from bone marrow macrophages (scale bar represents 500 μm). E , mRNA levels (qPCR assay) of osteoclast markers during osteoclastogenesis. F , representative pictures of Von Kossa-stained bone biomaterial surface (scale bar represents 500 μm) (n = 4/group). The resorbed areas appear white , and the unresorbed mineralized surface appears black . G , representative pictures of DAPI ( blue ; nuclei) and phalloidin ( red ; actin rings) staining of osteoclast cultures (scale bar represents 500 μm). H , mRNA levels (qPCR assay) of osteoclast fusion markers during osteoclastogenesis. All cultures were completed in triplicate. Lines and error bars represent mean ± SD. p Values were determined with ( A ) one-way ANOVA or ( C – H ) Student’s t test. DAPI, 4′,6-diamidino-2-phenylindole; Hem1, hematopoietic protein 1; M-CSF, hematopoietic protein 1; RANKL, receptor activator of nuclear factor-kappa B ligand; TRAP, tartrate-resistant acid phosphatase.

Article Snippet: Also, we used polyclonal antibodies for Hem1 (Novus Biologicals; catalog no.: NBP2-13643, 1/1000 dilution), c-Fos (Santa Cruz Biotechnology; catalog no.: sc-7202, 1/500 dilution), p38 (Cell Signaling; catalog no.: 9212, 1/1000 dilution), IκB (Santa Cruz Biotechnology; catalog no.: sc-847, 1/500 dilution), Akt (Cell Signaling; catalog no.: 9272, 1/1000 dilution), and p-CrkL (Cell Signaling Technology; catalog no.: 3181, 1/1000 dilution) to analyze their protein levels.

Techniques: Isolation, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Knock-Out, Generated, Staining

Journal: The Journal of Biological Chemistry

Article Title: Hematopoietic cytoplasmic adaptor protein Hem1 promotes osteoclast fusion and bone resorption in mice

doi: 10.1016/j.jbc.2022.102841

Figure Lengend Snippet: Rescue of cell fusion in Hem1-deficient osteoclasts by retroviral transduction of Hem1. Bone marrow macrophages lacking Hem1 were transduced with retroviral vectors expressing either empty vector (MSCV) or Hem1 (MSCV-Hem1) and were cultured with M-CSF and RANKL for ( A ) 24 h or ( B – D ) 5 days. A , protein levels (Western blot) of Hem1 in bone marrow macrophage cell cultures. B , representative pictures ( left ) and number ( right ) of TRAP-positive multinucleated osteoclasts with more than ten nuclei, generated from bone marrow macrophages (scale bar represents 500 μm). C , representative pictures ( left ) and resorbed areas ( right ) of Von Kossa-stained bone biomaterial surface (scale bar represents 500 μm) (n = 4/group). D , mRNA levels (quantitative PCR assay) of osteoclast markers in mOCs. All cultures were completed in triplicate. Lines and error bars represent mean ± SD. p Values were determined using Student’s t test. Hem1, hematopoietic protein 1; M-CSF, macrophage colony–stimulating factor; MSCV, murine stem cell virus; RANKL, receptor activator of nuclear factor-kappa B ligand; TRAP, tartrate-resistant acid phosphatase.

Article Snippet: Also, we used polyclonal antibodies for Hem1 (Novus Biologicals; catalog no.: NBP2-13643, 1/1000 dilution), c-Fos (Santa Cruz Biotechnology; catalog no.: sc-7202, 1/500 dilution), p38 (Cell Signaling; catalog no.: 9212, 1/1000 dilution), IκB (Santa Cruz Biotechnology; catalog no.: sc-847, 1/500 dilution), Akt (Cell Signaling; catalog no.: 9272, 1/1000 dilution), and p-CrkL (Cell Signaling Technology; catalog no.: 3181, 1/1000 dilution) to analyze their protein levels.

Techniques: Retroviral, Transduction, Expressing, Plasmid Preparation, Cell Culture, Western Blot, Generated, Staining, Real-time Polymerase Chain Reaction, Virus

Journal: The Journal of Biological Chemistry

Article Title: Hematopoietic cytoplasmic adaptor protein Hem1 promotes osteoclast fusion and bone resorption in mice

doi: 10.1016/j.jbc.2022.102841

Figure Lengend Snippet: Deletion of Hem1 attenuates respiration by suppressing c-Abl signaling. A – F , bone marrow macrophages from indicated genotypes were cultured with M-CSF and RANKL for 3 days. Different fractions of mitochondrial and nonmitochondrial respiration per 10,000 cells, in osteoclasts, were measured by Seahorse XF96 (n = 11–12 wells/group). G – I , bone marrow macrophages lacking Hem1 were cultured with M-CSF and RANKL for ( G ) 3 days or ( H and I ) indicated time points. G , ATP levels in osteoclasts (RLU, relative luminescence units; n = 4/group). H and I , protein levels (Western blot) in bone marrow macrophage cultures. To clarify Hem1 deletion in osteoclasts, the same images of Hem1 band are used. These are the same set of samples from the bone marrow macrophage cultures. J , osteoclasts developed in cultures of bone marrow macrophages from 6-month-old male C57BL/6 mice in the presence or the absence of imatinib (5 μM). Representative pictures ( left ), number ( middle ), and total area ( right ) of TRAP-positive multinucleated osteoclasts with more than ten nuclei (scale bar represents 500 μm) (triplicate cultures). K and L , bone marrow macrophages were isolated from 5.5-week-old male Hem1 knockout mice and wildtype littermates and were cultured with M-CSF and RANKL for ( K ) 5 or ( L ) 3 days in the presence or the absence of DPH (1 μM). K , number of TRAP-positive multinucleated osteoclasts generated from bone marrow macrophages. L , mRNA levels (quantitative PCR assay) of osteoclast markers during osteoclastogenesis. Lines and error bars represent mean ± SD. p Values were determined with ( A – G ) Student’s t test, ( J ) one-way ANOVA, or ( K and L ) two-way ANOVA. DPH, 5-(1, 3-diaryl-1H-pyrazol-4-yl); Hem1, hematopoietic protein 1; M-CSF, macrophage colony–stimulating factor; RANKL, receptor activator of nuclear factor-kappa B ligand; TRAP, tartrate-resistant acid phosphatase.

Article Snippet: Also, we used polyclonal antibodies for Hem1 (Novus Biologicals; catalog no.: NBP2-13643, 1/1000 dilution), c-Fos (Santa Cruz Biotechnology; catalog no.: sc-7202, 1/500 dilution), p38 (Cell Signaling; catalog no.: 9212, 1/1000 dilution), IκB (Santa Cruz Biotechnology; catalog no.: sc-847, 1/500 dilution), Akt (Cell Signaling; catalog no.: 9272, 1/1000 dilution), and p-CrkL (Cell Signaling Technology; catalog no.: 3181, 1/1000 dilution) to analyze their protein levels.

Techniques: Cell Culture, Western Blot, Isolation, Knock-Out, Generated, Real-time Polymerase Chain Reaction

Journal: The Journal of Biological Chemistry

Article Title: Hematopoietic cytoplasmic adaptor protein Hem1 promotes osteoclast fusion and bone resorption in mice

doi: 10.1016/j.jbc.2022.102841

Figure Lengend Snippet: Rescue of bone phenotype in Hem1-null mice by transplantation with wildtype hematopoietic cells. Hematopoietic stem cells from GFP + Lin − CD45 + wildtype mice efficiently repopulated the hematopoietic system of nonablated Hem1-deficient mice after bone marrow cell transplantation. Femurs and bone marrow were analyzed 10 weeks after transplantation (n = 5). A , schematic model of bone marrow transplantation. B , representative images of whole body ( left ) and longitudinal weight measurement ( right ). C , representative images of whole femur ( left ) and length measurement in 13-week-old male recipient Hem1 knockout mice and wildtype littermates ( right ). D , representative images of cortical bone at midshaft (scale bar represents 100 μm). E , cortical thickness and cortical perimeters at the midshaft. F , representative images of trabecular bone (scale bar represents 1 mm) and ( G ) bone volume per tissue volume (BV/TV), bone mineral density (BMD), and microarchitecture of trabecular bone. H , serum concentration of a collagen degradation product (CTx) by ELISA. I , representative pictures ( left ) and resorbed areas ( right ) of Von Kossa-stained bone biomaterial surface (scale bar represents 500 μm) (triplicate cultures). J – M , bone marrow macrophages were isolated from 13-week-old recipient Hem1 knockout mice and wildtype littermates and cultured with M-CSF and RANKL for ( G , I , and J ) 5 days or ( K and M ) 3 days. J , representative pictures ( left ) and number ( right ) of TRAP-positive multinucleated osteoclasts with more than ten nuclei (scale bar represents 500 μm) (n = 6). K , mRNA levels (quantitative PCR assay) of osteoclast markers during osteoclastogenesis (triplicate cultures). L , representative pictures of DAPI ( blue , nuclei) and phalloidin ( red , actin rings) staining in osteoclast cultures (scale bar represents 500 μm). M , mRNA levels (quantitative PCR assay) of fusion markers during osteoclastogenesis (triplicate cultures). Lines and error bars represent mean ± SD. p Values were determined with Student’s t test. DAPI, 4′,6-diamidino-2-phenylindole; Hem1, hematopoietic protein 1; M-CSF, hematopoietic protein 1; RANKL, receptor activator of nuclear factor-kappa B ligand; TRAP, tartrate-resistant acid phosphatase.

Article Snippet: Also, we used polyclonal antibodies for Hem1 (Novus Biologicals; catalog no.: NBP2-13643, 1/1000 dilution), c-Fos (Santa Cruz Biotechnology; catalog no.: sc-7202, 1/500 dilution), p38 (Cell Signaling; catalog no.: 9212, 1/1000 dilution), IκB (Santa Cruz Biotechnology; catalog no.: sc-847, 1/500 dilution), Akt (Cell Signaling; catalog no.: 9272, 1/1000 dilution), and p-CrkL (Cell Signaling Technology; catalog no.: 3181, 1/1000 dilution) to analyze their protein levels.

Techniques: Transplantation Assay, Knock-Out, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Isolation, Cell Culture, Real-time Polymerase Chain Reaction