Display Settings:

Items per page

Results: 7

1.
Fig 4

Fig 4. Myoblasts from the α-syntrophin-knockout mice do not respond to HGF stimulation. From: ?-Syntrophin is required for the hepatocyte growth factor-induced migration of cultured myoblasts.

Skeletal myoblasts were isolated from C57, α-syntrophin-knockout (αKO), α/β2-syntrophin-double knockout (AB), and α-syntrophin transgenic (FLA) mice and seeded on the insert of a three dimensional chamber assay as described in “Materials and Methods”. After incubation for 1 h, cells were fixed with cold methanol and stained with hematoxylin. The cells migrating to the lower chamber were counted using an inverted microscope. Values are expressed as means ± S.E.M. from four independent experiments (*p<0.01, **p<0.001).

Min Jeong Kim, et al. Exp Cell Res. ;317(20):2914-2924.
2.
Fig 7

Fig 7. Localization of PTEN is disrupted in the α-syntrophin knock-down cells. From: ?-Syntrophin is required for the hepatocyte growth factor-induced migration of cultured myoblasts.

Cells grown on a 24-well tissue culture plates for 24 h were transfected with control siRNA or α-syntrophin-specific siRNA (100 nM) for 6 h. Cells were then incubated with (+) or without (−) HGF (50 ng/ml) for 1 h and were labeled with anti-α-syntrophin and anti-PTEN antibodies. Dotted line indicates shape of the cell. Small arrows indicate the lamellipodia of cells and the orange arrow indicates the direction of cell migration. Asterisks indicate co-localization of α-syntrophin and PTEN in the rear-lateral part of the cell. The scale bar is 10 μm.

Min Jeong Kim, et al. Exp Cell Res. ;317(20):2914-2924.
3.
Fig 6

Fig 6. Akt phosphorylation is reduced in the α-syntrophin-specific siRNA transfected cells. From: ?-Syntrophin is required for the hepatocyte growth factor-induced migration of cultured myoblasts.

(A) C2 cells were transfected with control siRNA (lane C) or increasing concentration of α-syntrophin-specific siRNA and treated with HGF for 1 h. The cells were harvested and the levels of α-syntrophin, Akt, and phosphor-Akt (pAkt) were determined by western blotting. Actin was used as a loading control. (B) Cells transfected with control siRNA or α-syntrophin-specific siRNA (100 nM) were treated with (+) or without HGF (−) for 1 h. Akt phosphorylation (S473 residue) was determined by western blotting. The intensity of phosphor-Akt band was measured with Scion image software. The level of the phosphor-Akt in control siRNA-transfected cells without HGF is expressed as 1.0. Values are expressed as means ± S.E.M. from four independent experiments (*p<0.05).

Min Jeong Kim, et al. Exp Cell Res. ;317(20):2914-2924.
4.
Fig 1

Fig 1. HGF induces lamellipodia formation of C2 myoblasts. From: ?-Syntrophin is required for the hepatocyte growth factor-induced migration of cultured myoblasts.

(A) C2 myoblasts cultured on cover-slips for 6 h in growth media were transferred into serum-starved DMEM, pre-incubated for 1 h, then treated with HGF (50 ng/ml) for 1 h. The formation of lamellipodia (arrows) detected by labeling with rhodamine-phalloidin (red). Orange arrow indicates the direction of cell migration. Nuclei were stained with DAPI (blue). Scale bar is 10 μm. (B) Under the same conditions, cells were incubated with (+) or without (−) HGF (50 ng/ml) for the indicated times (0 min is the time point of HGF treatment). Cells with lamellipodia were counted from 20 randomly selected fields and the formation of lamellipodia was expressed as a ratio to the total nuclei in the fields (*p < 0.001).

Min Jeong Kim, et al. Exp Cell Res. ;317(20):2914-2924.
5.
Fig 5

Fig 5. Inhibitors of PI3-kinase block the HGF-induced migration of C2 cells. From: ?-Syntrophin is required for the hepatocyte growth factor-induced migration of cultured myoblasts.

(A) C2 myoblasts cultured on coverslips were treated with LY294002 (20 μM) or Wortmannin (0.5 μM) for 30 min. Then the cells were incubated with (+) or without (−) HGF for 1 h. Migrating cells were observed by staining with rhodamine-phalloidin. Cells were counted from 20 randomly selected fields (*p<0.001). (B) Under the same culture conditions, cells were labeled with BSA solution containing the anti-pan-syntrophin or anti-PTEN antibodies. Then cells were incubated with fluorescence-conjugated secondary antibodies and DAPI for 40 min at room temperature. The specimens were observed with a Zeiss LSM 510 confocal laser scanning microscope. Asterisks indicate co-localization of the two proteins. Orange arrow indicates the direction of cell migration. Bar indicates 10 μm.

Min Jeong Kim, et al. Exp Cell Res. ;317(20):2914-2924.
6.
Fig 3

Fig 3. α-Syntrophin siRNA-treated C2 cells do not respond to HGF stimulation. From: ?-Syntrophin is required for the hepatocyte growth factor-induced migration of cultured myoblasts.

(A) Cells were cultured on a 24-well tissue culture plate for 24 h and transfected with control siRNA or α-syntrophin-specific siRNA (100 nM) for 6 h. Cells were then treated with HGF (50 ng/ml) for 1 h and were labeled with rhodamine-phalloidin (red) and anti-α-syntrophin antibody. Arrows indicate lamellipodia and the asterisk shows α-syntrophin in the rear-lateral part of the cell. Big arrow in orange color indicates the direction of migrating cell. Scale bar is 10 μm. (B) Under identical culture conditions, the cells with lamellipodia were observed using a fluorescence microscopy. Cells were counted from 20 randomly selected fields. (*p<0.001). Cell lysates were subjected to western blot to determine protein level of α-syntrophin after siRNA transfection. Actin is used as a loading control.

Min Jeong Kim, et al. Exp Cell Res. ;317(20):2914-2924.
7.
Fig 2

Fig 2. Syntrophins are localized at the trailing edge of the migrating cells. From: ?-Syntrophin is required for the hepatocyte growth factor-induced migration of cultured myoblasts.

(A) C2 myoblasts on coverslips were prepared as described in Fig 1. Cells were incubated with (+) or without (−) HGF (50 ng/ml) for 1 h and then labeled with anti-pan-syntrophin, anti-PTEN, or anti-p85 antibodies. Arrowheads and asterisks indicate syntrophin and PTEN in rear-lateral part of the cell, respectively. Arrows show p85 in lamellipodia of the cell. PTEN is used as a marker for the trailing edge whereas p85 is used for the lamellipodia at the leading edge of migrating cells. Rhodamine-phalloidin was used to stain F-actin (red). Fluorescence images were taken on a Zeiss LSM 510 confocal laser scanning microscope or Zeiss Axioskop 40 FL microscope. The scale bar indicates 10 μm. (B) Under the same culture conditions, cells were co-labeled with anti-pan-syntrophin and anti-PTEN antibodies, or anti-pan-syntrophin and anti-p85 antibodies. Arrows indicate co-localization of syntrophin and PTEN. Asterisks indicate p85 in lamellipodia. Bar is 10 μm. (C) C2 cells transfected with GFP fusion α-syntrophin were incubated with or without HGF (50 ng/ml). An arrow indicates GFP fusion α-syntrophin in rear-lateral part of the cell. To visualize the cytoskeleton and nuclei, cells were labeled with rhodamine-phalloidin (red) and DAPI, respectively. The scale bar indicates 10 μm. Orange arrow indicates the direction of cell migration. (D) Cells cultured as above were harvested for immunoprecipitation with anti-PTEN or anti-pan-syntrophin antibodies. The immunoprecipitated proteins were separated on SDS-PAGE and subjected to western blot analysis with anti-α-syntrophin or anti-PTEN antibodies. Lane ‘Total’ shows the western blot of the protein extract prior to immunoprecipitation and indicates the size of the expected proteins.

Min Jeong Kim, et al. Exp Cell Res. ;317(20):2914-2924.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk