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Results: 5

1.
Fig. 5

Fig. 5. Src, GIT1 and PLCγ2 localizes in the podosome belts. From: GPCR kinase 2 interacting protein 1 (GIT1) regulates osteoclast function and bone mass.

WT KO BM cells were differentiated into OC by treatment with MCSF (20 ng/ml) and RANKL (50 ng/ml) for 7 days, fixed and stained for (A) Src, (B) GIT1 and (C) PLCγ2. (D) WT OCs were serum starved for 4 hrs and stimulated with RANKL (100ng/ml) for 5 mins, fixed and stained for phosphorylated form of PLCγ2 using phospho- PLCγ2 (Y759) antibody. Rhodamine-phalloidin was used to visualize actin rings. (Scale bar: 15 μm)

Prashanthi Menon, et al. J Cell Physiol. ;225(3):777-785.
2.
Fig. 2

Fig. 2. GIT1 KO mice have normal osteoclast number. From: GPCR kinase 2 interacting protein 1 (GIT1) regulates osteoclast function and bone mass.

(A) BM cells from GIT1 WT were differentiated into OC with MCSF (20 ng/ml) and RANKL (50 ng/ml). Cell lysates were harvested on day 7 and protein expression probed using GIT1 antibody. GIT1 KO OC did not show GIT1 protein expression. Actin serves as loading control. (B,C) TRAP stain in the femur sections of GIT1 WT and KO mice. OC per mm2 were quantified using light microscopy. WT= 10±1; KO= 11± 2. Values are expressed as mean ± SEM (n=3) Scale bar: 50μm. (D,E) GIT1 WT and KO femur sections were stained with alcian blue to visualize cartilage and counterstained with Orange O-eosin for bone. Note increased presence of cartilage remnants (blue) in the trabecular bone (orange) in GIT1 KO mice (E, arrows) (Scale bar: 20 μm, n=3).

Prashanthi Menon, et al. J Cell Physiol. ;225(3):777-785.
3.
Fig. 4

Fig. 4. GIT1 functions downstream of RANK signaling. From: GPCR kinase 2 interacting protein 1 (GIT1) regulates osteoclast function and bone mass.

(A) GIT1 WT and KO BM cells were differentiated into OC by treatment with RANKL (50 ng/ml) and MCSF (20 ng/ml) for 7 days. Cells were serum starved for 6 hrs, stimulated with 100 ng/ml RANK for the indicated times. Cell lysates were harvested and probed with 4G10 phosphotyrosine antibody to visualize GIT1 phosphorylation (97 kd, arrow). Blots were then probed for GIT1 expression. (B) WT BM OC were treated with Src inhibitor PP2 (10 μM for 1 hr), stimulated with 100 ng/ml RANKL for 5 mins. GIT1 phosphorylation was probed using 4G10 antibody. Blots were reprobed for GIT1 expression. (C) GIT1 WT and KO OC on day 7 were starved and treated with RANKL (100 ng/ml) for indicated times. Lysates were immunoblotted with phoshorylated specific PLCγ2 (Y759) antibody (p-PLCγ2 Y759). Fold induction of normalized, phosphorylated protein vs time 0 of the control are shown.

Prashanthi Menon, et al. J Cell Physiol. ;225(3):777-785.
4.
Fig. 1

Fig. 1. GIT1 KO mice have increased bone mass. From: GPCR kinase 2 interacting protein 1 (GIT1) regulates osteoclast function and bone mass.

(A) Three dimensional microCT analysis of the long bones from 10-12 week old sex matched GIT1 WT and KO mice. Wt n=9; KO n=8. (B-E) Bone morphometric analysis of the long bones from GIT1 WT and KO mice. BV/TV%: percentage of bone volume (BV)/total volume (TV); Conn. Density (1/mm3): Connectivity density per mm3. All values are expressed as mean ± SEM. Data analysis was done by two-tailed unpaired student’s t-test. A p<0.05 was considered to statistically significant. (F) Longitudinal sections of femur bones of GIT1 WT and KO mice stained with H&E. Trabecular bone number and connectivity are greater in GIT1 KO femurs compared to WT controls as indicated by arrows. (Scale bar: 20 μm) n=3 per group.

Prashanthi Menon, et al. J Cell Physiol. ;225(3):777-785.
5.
Fig. 3

Fig. 3. GIT1 deficiency impairs osteoclast function. From: GPCR kinase 2 interacting protein 1 (GIT1) regulates osteoclast function and bone mass.

GIT1 WT and KO BM cells were differentiated into OC for 7 days on (A,B) culture dish and (C,D) dentin slices. Cells were fixed, TRAP stained and TRAP positive cells greater than 3 nuclei (arrows) were quantified using a light microscope (WT= 80±10, KO=75 ±12, p>0.05). (E, F) Resorbtion pits on dentine slices were examined by toludiene stain after removing OCs. (G) Quantitative analysis of the pit area volume as percentage of control showed a 65% decrease in GIT1 KO OC. (H, I) GIT1 WT and KO OC were fixed on day 7 and stained for F-actin rings using rhodamine phalloidin. In WT OC, F-actin (red) was organized in podosome belts (H) while GIT1 KO OC showed loss of podosome belts (I). Nuclei (blue) were stained with DAPI to identify multinucleated cells. (Scale bar: 15 μm) (J) Quantitative analysis of number of normal podosome belts in GIT1 WT and KO OC showed a 60% decrease in GIT1 KO OC. A total of 70 OC were counted from three individual experiments. All values are expressed as mean ± SEM. (n=3). Data analysis was done by two-tailed unpaired student’s t-test. A p<0.05 was considered statistically significant.

Prashanthi Menon, et al. J Cell Physiol. ;225(3):777-785.

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