Reduction of the down-regulation of Necl-5 by knockdown of nectin-3. NIH3T3 cells were transfected with the synthetic siRNA against nectin-3 or the control synthetic siRNA and cultured for 48 h. The transfection efficiency was >90%. (A) Confirmation of the knockdown of nectin-3. (a) Western blotting. The cell lysates were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–nectin-3 pAb. (b and c) Immunofluorescence images. The cells were double stained with the anti–nectin-3 mAb and the anti–N-cadherin pAb. (b) Control; (c) nectin-3 siRNA. Bars, 10 μm. (B) Reduction of the down-regulation of Necl-5 by knockdown of nectin-3. The siRNA-transfected cells were cultured at the lower or higher density. (a) Western blotting of cell surface proteins. Biotinylated cell surface proteins were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 mAb-i, the anti–nectin-1 pAb, the anti–nectin-3 pAb, or the anti–N-cadherin mAb. (b and c) Immunofluorescence images. The cells were double stained with the anti–Necl-5 mAb-i and the anti–nectin-3 pAb. (b) Control; (c) nectin-3 siRNA. Bars, 10 μm. The results shown are representative of three independent experiments.

Clathrin-dependent endocytosis of Necl-5. (A) Inhibition of down-regulation of Necl-5 by a DN mutant of epsin, Eps15, or dynamin, but not by a DN mutant of caveolin. Immunofluorescence images of the cells transfected with various DN mutants. The cells were transfected with myc-epsin DN mutant (a), EGFP-Eps15 DN mutant (b), HA-dynamin DN mutant (c), or EGFP-caveolin DN mutant (d); cultured at the higher density; and double or triple stained with various combinations of the anti–Necl-5 mAb-i, the anti-myc mAb, the anti-HA mAb, and the anti–nectin-3 pAb. Asterisks show the transfected cells. Bars, 10 μm. (B) Assembly of clathrin heavy chain at the cytoplasmic region of Necl-5, which interacts with Nef-3. NIH3T3 cells were cultured at the lower density and then cultured in the presence or absence of Nef-3– or IgG-coated beads for 1 h. The cells were double stained with the anti–Necl-5 mAb-i and the anticlathrin heavy chain mAb. (a) In the presence of the Nef-3–coated beads; (b) in the presence of the IgG-coated beads; (c) in the absence of the beads. Asterisks, the positions of the beads; insets, magnified images of the boxed areas. Bars, 10 μm. The results shown are representative of three independent experiments.

Inhibition of cell density–dependent reduction of cell movement and DNA synthesis by the knockdown of nectin-3. NIH3T3 cells were transfected with the synthetic siRNA against nectin-3 or the control synthetic siRNA and cultured for 48 h. The knockdown of nectin-3 inhibited the cell density–dependent down-regulation of Necl-5 (see Fig. 2). (A) Measurement of cell movement by Boyden chamber assay. The transfected cells were cultured at the lower or higher density for 24 h, replated in culture insert, and cultured for 9 h. The migrated cells were counted. *, P < 0.001. (B) Measurement of DNA synthesis by BrdU incorporation. The transfected cells were cultured at the lower or higher density for 24 h, incubated with BrdU for 2 h, and then double stained with the anti-BrdU mAb and DAPI. (a and c) Control; (b and d) nectin-3 siRNA; (e) quantification of BrdU-positive cells. Bars, 20 μm. *, P < 0.001. The results are the mean ± SEM of the three independent experiments.

Cell density–dependent down-regulation of Necl-5. (A) Cell density–dependent down-regulation of Necl-5. NIH3T3 cells were seeded, starved of serum, and cultured in the medium containing serum in the presence or absence of the anti–Necl-5 mAb-i. The cells were sampled at each indicated time. (a) Cell growth curves, amounts of cell surface Necl-5, and levels of Necl-5 mRNA. The relative amounts of cell surface Necl-5 and the level of Necl-5 mRNA at 0 h were set to 1. (b) Western blotting of cell surface proteins. Biotinylated cell surface proteins were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 pAb, the anti–nectin-1 pAb, the anti–nectin-3 pAb, or the anti–N-cadherin mAb. (B–E) Down-regulation of Necl-5 in the cells cultured at the higher density. NIH3T3 cells were cultured at the lower or higher density for 24 h. (B) Western blotting of cell surface proteins in the absence or presence of the anti–Necl-5 mAb-i. Biotinylated cell surface proteins were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 pAb, the anti–nectin-1 pAb, the anti–nectin-3 pAb, or the anti–N-cadherin mAb. (C) FACS analysis of cell surface Necl-5. The cells were stained with the anti–Necl-5 mAb-i. (D) Immunofluorescence images. The cells were double stained with various combinations of the anti–Necl-5 mAb-i, the anti–nectin-3 mAb, the anti–N-cadherin pAb, and the antiactin mAb. Bars, 10 μm. (E) The levels of Necl-5 mRNA. The level of Necl-5 mRNA at the lower cell density was set to 1. The results shown in A and E are the mean ± SEM of the three independent experiments, and the results shown in B, C, and D are representative of three independent experiments.

Down-regulation of Necl-5 by its interaction with nectin-3. NIH3T3 cells were cultured at the lower density in the absence or presence of Nef-3, other recombinant proteins, and/or mAb for 1 h. (A) Immunofluorescence images. The cells were single or double stained with various combinations of the anti–Necl-5 mAb-i, the rhodamine-conjugated anti–human IgG Fc pAb, and the anti-Rab7 pAb. (a) Control; (b, f, and g) in the presence of 0.3 μM Nef-3; (c) in the presence of 0.3 μM Nef-1; (d) in the presence of 0.3 μM Nef-3 and 50 μg/ml of the anti–Necl-5 mAb-i; (e) in the presence of 0.3 μM Nef-3 and Nef-1. (a–e and g) Immunostaining with permeabilization; (f) immunostaining without permeabilization. Arrowheads show the signal for Necl-5 at the leading edges of the cells. Bars, 10 μm. (B) Western blotting of cell surface proteins in the absence or presence of 0.3 μM Nef-3. Biotinylated cell surface proteins were subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 pAb or the anti–N-cadherin mAb. (C) FACS analysis of cell surface Necl-5 in the absence or presence of 0.3 μM Nef-3. The cells were fixed with 1% formaldehyde before FACS analysis and stained with the anti–Necl-5 mAb-i. (D) Western blotting of endocytosed Necl-5 in the absence or presence of 0.3 μM Nef-3. Necl-5, which was biotinylated and endocytosed, was subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 pAb. The results shown are representative of three independent experiments.

Cell density–dependent reduction of cell movement and DNA synthesis by the down-regulation of Necl-5. (A) Measurement of cell movement by Boyden chamber assay. (a) Quantification of migrated cells. NIH3T3 cells were cultured at the lower or higher density in the presence or absence of the anti–Necl-5 mAb-i for 24 h, replated in culture insert, and cultured for 9 h. The migrated cells were counted. *, P < 0.001. (b) Reversion of the level of cell surface Necl-5. The cells were cultured at the lower or higher density in the presence or absence of the anti–Necl-5 mAb-i, replated at the lower density, and cultured for 9 h. Biotinylated cell surface Necl-5 was collected before (0 h) or after (9 h) replating and subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 mAb-i. (B) Measurement of DNA synthesis by BrdU incorporation. The cells were cultured at the lower or higher density in the presence or absence of the anti–Necl-5 mAb-i for 24 h, incubated with BrdU for 2 h, and then double stained with the anti-BrdU mAb and DAPI. (a and c) In the absence of the anti–Necl-5 mAb-i; (b and d) in the presence of the anti–Necl-5 mAb-i; (e) quantification of BrdU-positive cells. Bars, 20 μm. *, P < 0.001. The results are the mean ± SEM of the three independent experiments.

Reduction of cell movement and DNA synthesis by the knockdown of Necl-5. NIH3T3 cells were transfected either with the siRNA vector against Necl-5 and the pEGFP-tub vector or with the control siRNA vector and the pEGFP-tub vector and cultured at the lower density for 48 h. The transfection efficiency was ∼20%. (A) Confirmation of the knockdown of Necl-5. (a) FACS analysis. The cells were stained with the anti–Necl-5 mAb-i and the EGFP-tub–positive cells were monitored. (b) Western blotting. The EGFP-tub–positive cells were sorted by FACS and then the lysate of the sorted cells was subjected to SDS-PAGE (10% polyacrylamide gel), followed by Western blotting with the anti–Necl-5 mAb-i. (c) Immunofluorescence images. The cells were single stained with the anti–Necl-5 mAb-i. (c1) Necl-5; (c2) Necl-5 and EGFP-tub. Asterisks show the Necl-5 knockdown cells. Bar, 10 μm. (B) Measurement of cell movement by Boyden chamber assay. The transfected cells (∼20% transfection efficiency) were cultured at the lower density, directly replated in culture insert, and cultured for 9 h. The EGFP-tub–positive migrated cells were counted. The number of control EGFP-tub–positive migrated cells was consistent with that of control migrated cells precultured at lower density in Fig. 5 A (a) and Fig. 6 A, considering the transfection efficiency. *, P < 0.001. (C) Measurement of DNA synthesis by BrdU incorporation. The transfected cells (∼20% transfection efficiency) were directly cultured at the lower density for 18 h and incubated with BrdU for 2 h. The cells were double stained with the anti-BrdU mAb and DAPI. The EGFP-tub–positive cells were measured. (a) Control; (b) Necl-5 siRNA; (c) quantification of BrdU-positive cells. Bars, 20 μm. *, P < 0.001. The results shown in A are representative of three independent experiments, and the results shown in B and C are the mean ± SEM of the three independent experiments.