(A) Untransfected U2OS cells or U2OS cells stably expressing FGFR1 or FGFR3 were incubated for 20 minutes at 37°C with Cy3-FGF1 and 50 U/ml heparin. The cells were stained with Hoechst 33342 and examined with confocal microscopy. Bar, 5 µm. (B) U2OS cells stably expressing FGFR1 or FGFR3 were grown on gelatinized plates and incubated with 10 ng/ml ¹²⁵I-FGF1, 20 U/ml heparin and 0.2% gelatine at 37°C for indicated periods of time. Internalized and surface-bound ¹²⁵I-FGF1 were separated as described in Materials and methods and the ratio was plotted as function of time. The graph represents the mean ±s.d. of three independent experiments with three parallels. (B) U2OS cells stably expressing FGFR1 or FGFR3 were subjected to vector-based siRNA against CHC for 4 days to knock down clathrin. The cells were then incubated for 20 minutes at 37°C with Cy3-FGF1, Alexa 647-Tf and 50 U/ml heparin. The cells were stained with goat anti-CHC antibody and examined with confocal microscopy. Bar, 5 µm. (C) The uptake of Cy3-FGF1 was measured as Cy3 intensity in non-depleted and CHC depleted cells treated as described in B. Only cells in which Tf uptake was inhibited were defined as depleted of CHC. The mean intensity of Cy3 in CHC depleted cells is presented in the histogram as the percentage of Cy3 intensity in non-depleted cells. The histogram represents the mean +s.d. of three independent experiments and 15-45 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings. (D) U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos (100 nM) targeting CHC or a non-targeting siRNA control (scr) as described in Materials and Methods. The level of knockdown was assessed in every experiment by Western blotting using mouse anti-CHC antibody. Anti-Hsp90 antibody was used as a loading control. One representative Western blot is shown. (E) U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos (100 nM) targeting CHC or a non-targeting siRNA control (scr), grown on gelatinized plates and incubated with 10 ng/ml ¹²⁵I-FGF1, 20 U/ml heparin and 0.2% gelatine at 37°C for indicated periods of time. Internalized and surface-bound ¹²⁵I-FGF1 were separated as described in Materials and Methods and the ratio was plotted as function of time. Note the different scale on the Y axis. The graph represents the mean ±s.d. of three independent experiments with three parallels.

U2OS cells stably transfected with FGFR1 (A) or FGFR3 (B) were transfected with HA-tagged dynamin constructs as indicated and incubated with Cy3-FGF1 and 50 U/ml heparin at 37°C for 20 min. The cells were then fixed and stained with anti-HA antibody. The cells were examined with confocal microscopy. Bar, 5 µm.

(A) U2OS cells stably transfected with FGFR1 or FGFR3 were transfected with HA-tagged Arf6 T27N or EGFP-Cdc42 N17 and incubated with Cy3-FGF1 and 50 U/ml heparin at 37°C for 20 min. The cells were then fixed and examined with confocal microscopy. Cells transfected with Arf6 T27N were stained with anti-HA antibody. Bar, 5 µm. (B) The uptake of Cy3-FGF1 was measured as Cy3 intensity in untransfected or Arf6 T27N or Cdc42 N17 transfected cells. The mean intensities of Cy3 in transfected cells are presented in the histograms as the percentage of Cy3 intensity in untransfected cells. The histogram represents the mean +s.d. of three independent experiments and 15–55 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings. U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos (25 nM) targeting Arf6 (C) or Cdc42 (E) or a non-targeting siRNA control (scr) as described in Materials and Methods. The level of knockdown was assessed by Western blotting using rabbit anti-Arf6 antibody or rabbit anti-Cdc42 antibody. Anti-Hsp90 antibody was used as a loading control. One representative Western blot is shown. Western blots were quantified and the bands corresponding to Arf6 and Cdc42 were normalized to loading control and knockdown efficiency presented in the histogram as percentage of non-targeting siRNA control (scr). The histogram represents the mean +s.d. of three independent experiments. U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos (25 nM) targeting Arf6 (D) or Cdc42 (F) or a non-targeting siRNA control (scr), grown on gelatinized plates and incubated with 10 ng/ml ¹²⁵I-FGF1, 20 U/ml heparin and 0.2% gelatine at 37°C for indicated periods of time. Internalized and surface-bound ¹²⁵I-FGF1 were separated as described in Materials and Methods and the ratio was plotted as function of time. Note the different scale on the Y axis. The graph represents the mean ±s.d. of three independent experiments with three parallels.

U2OS cells stably expressing FGFR1 (A) or FGFR3 (B) were transfected with siRNA oligos (100 nM) targeting CHC or a non-targeting siRNA control (scr), serum-starved for 4–6 hours and then stimulated with the growth factor in the presence of heparin (20 U/ml) and cycloheximide (10 µg/ml) for different time points. Cells were lysed, and the cellular material was analyzed by SDS-PAGE and immunoblotting using following antibodies: anti CHC, anti-FGFR1/anti-FGFR3, anti-phospho-FGFR (p-FGFR), anti-phospho-FRS2α (p-FRS2α), anti-phospho-MAPK (p-MAPK) and anti-Hsp90 as a loading control. (C) The amount of FGFR, p-FGFR, p-FRS2α and p-MAPK were expressed as a percentage with the maximum for each protein set to 1. The graph represents the mean ±s.d. of three independent experiments.

U2OS cells stably expressing FGFR1 (A) or FGFR3 (B) were transfected with siRNA oligos (100 nM) targeting CHC or a non-targeting siRNA control (scr), grown on gelatinized plates and incubated with 2, 20 or 100 ng/ml ¹²⁵I-FGF1, 20 U/ml heparin and 0.2% gelatine at 37°C for indicated times. Internalized and surface-bound ¹²⁵I-FGF1 were separated as described in Materials and Methods and the ratio was plotted as function of time. Note the different scale on the Y axis. The graph represents one independent experiment with three parallels ±s.d.

U2OS cells stably transfected with FGFR1 (A) or FGFR3 (B) were transfected with HA-tagged dynamin 1 K44A and incubated with Cy3-FGF1 and 50 U/ml heparin at 37°C for 20 min. The cells were then fixed and stained with anti-HA and anti-EEA1 antibodies. In some cases the cells were washed with high salt/low pH buffer (HSLP) to remove surface-bound Cy3-FGF1 before fixation. The cells were examined with confocal microscopy. Arrows point to cells transfected with dynamin 1 K44A. Selected areas are enlarged and shown at the right side. Bar, 5 µm. (C) The uptake of Cy3-FGF1 was measured as Cy3 intensity in untransfected and dynamin 1 K44A transfected cells. All cells were washed with HSLP-buffer to remove cell-surface bound Cy3-FGF1. The mean intensity of Cy3 in dynamin 1 K44A transfected cells is presented in the histogram as the percentage of Cy3 intensity in untransfected cells. The histogram represents the mean +s.d. of three independent experiments and 20–60 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings.

(A) U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos targeting flotillin 1 and flotillin 2 or a non-targeting siRNA control (scr) as described in Materials and Methods. The level of knockdown was assessed in every experiment by Western blotting using mouse anti-flotillin 1 and mouse anti-flotillin 2 antibodies. Anti-γ-tubulin antibody was used as a loading control. One representative Western blot is shown. The bands corresponding to flotillin 1 and flotillin 2 were quantified and knockdown efficiency is presented in the histogram as percentage of non-targeting siRNA control (scr). The histogram represents the mean +s.d. of 12 (for flotillin 1) and 7 (for flotillin 2) independent experiments. (B) U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos targeting flotillin 1 and 2 or a non-targeting siRNA control (scr). The cells were then incubated for 20 minutes at 37°C with Cy3-FGF1 and 50 U/ml heparin. The cells were stained with rabbit anti-flotillin 1 antibody or rabbit anti-flotillin 2 antibody and examined with confocal microscopy. Bar, 5 µm. (C) The uptake of Cy3-FGF1 was measured as Cy3 intensity in non-depleted and flotillin 1 and 2 depleted cells treated as described in B. The mean intensity of Cy3 in flotillin 1 and 2 depleted cells is presented in the histogram as the percentage of Cy3 intensity in non-depleted cells. The histogram represents the mean +s.d. of four independent experiments and 67–138 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings. (D) U2OS cells stably transfected with FGFR1 or FGFR3 were co-transfected with HA-tagged Arf6 T27N and EGFP-Cdc42 N17 and incubated with Cy3-FGF1 and 50 U/ml heparin at 37°C for 20 min. The cells were then fixed, stained with anti-HA antibody and examined with confocal microscopy. Bar, 5 µm. (E) U2OS cells stably expressing FGFR1 or FGFR3 were transfected with siRNA oligos targeting flotillin 1 and 2 or a non-targeting siRNA control (scr). The cells were then transfected with HA-tagged Arf6 T27N and EGFP-Cdc42 N17 and incubated for 20 minutes at 37°C with Cy3-FGF1 and 50 U/ml heparin. The cells were fixed, stained with anti-HA antibody and examined with confocal microscopy. Bar, 5 µm. (F) The uptake of Cy3-FGF1 was measured as Cy3 intensity in untransfected or co-transfected cells (Arf6 T27N and Cdc42 N17) or in the case of flotillin knockdown as untransfected scr cells or co-transfected (Arf6 T27N and Cdc42 N17), flotillin1/2 siRNA cells. The mean intensities of Cy3 in transfected cells are presented in the histograms as the percentage of Cy3 intensity in untransfected cells. The histogram represents the mean +s.d. of three independent experiments and 32–52 cells were quantified in each case in each experiment. Confocal scanning was performed with identical settings.