Figure 4. Treatment of MCF-7 cells with... | Rockefeller University Press

Figure 4.

$Figure 4. Treatment of MCF-7 cells with a GrB inhibitor reduced nuclear FGFR1 accumulation and blocked FGF-10–dependent cell migration. (A) Treatment of MCF-7 cells, growing in serum-containing medium, with a GrB inhibitor (inh.) abolished nuclear FGFR1 (arrows) after 24 h but did not change the total FGFR1 protein levels. *, P < 0.05 (ANOVA). Bar, 100 µm. (B) Subcellular fractionation confirmed that FGF-10 treatment (1–2 h) increased nuclear FGFR1 (a truncated 55-kD C-terminal fragment of FGFR1) in serum-starved MCF-7 cells and decreased it when cells were treated with 25 µM GrB inhibitor (24 h). When cells were pretreated with GrB inhibitor and then treated with FGF-10, the translocation of the nuclear FGFR1 was partially blocked. Immunoblotting with Lamin A/C and tubulin antibodies confirmed the specificity of the cell fractionation protocol. Nuclear FGFR1 was normalized to the nuclear marker Lamin A/C, confirming the significance of the accumulation (graph). (C) MCF-7 cells were treated with 25 µM GrB inhibitor (24 h) in serum-free medium before stimulation with FGF-10/heparin (100 ng/ml and 7.5 µg/ml, respectively). Control cells showed increased staining for nuclear FGFR1 (arrows) after FGF-10 treatment, and this was blocked by GrB inhibition. Bar, 25 µm. (D) Transwell migration assays with MCF-7 cells showed that GrB inhibition abolished the promigratory effect of FGF-10. In contrast, cells were still able to migrate in response to EGF stimulation. *, P ≤ 0.05; **, P ≤ 0.01 (Student’s t test). (E) GrB activity in lysates from serum-starved MCF-7 cells with or without FGF-10/heparin treatment (100 ng/ml and 7.5 µg/ml, respectively) in the presence or absence of pretreatment with the serine protease inhibitor 3,4-dichloroisocoumarin (DCI; 50 µM; 1-h pretreatment). Activity was measured at 405-nm absorbance at hourly intervals after the addition of 200 µM Ac-IEPD-pNA substrate. FGF-10 treatment significantly increased GrB activity in MCF-7 cells, but this effect was abrogated by pretreatment with DCI. **, P ≤ 0.01; ***, P ≤ 0.001 (Student’s t test). (F) GrB activity in MCF-7 cells growing in full serum is markedly reduced by treatment with 50 µM DCI (2 h). (G) 5-h treatment with 50 µM DCI decreased nuclear FGFR1 protein significantly. *, P ≤ 0.05 (Student’s t test). Error bars show means ± SEM. A.U., arbitrary unit; Ctr, control.$

Treatment of MCF-7 cells with a GrB inhibitor reduced nuclear FGFR1 accumulation and blocked FGF-10–dependent cell migration. (A) Treatment of MCF-7 cells, growing in serum-containing medium, with a GrB inhibitor (inh.) abolished nuclear FGFR1 (arrows) after 24 h but did not change the total FGFR1 protein levels. *, P < 0.05 (ANOVA). Bar, 100 µm. (B) Subcellular fractionation confirmed that FGF-10 treatment (1–2 h) increased nuclear FGFR1 (a truncated 55-kD C-terminal fragment of FGFR1) in serum-starved MCF-7 cells and decreased it when cells were treated with 25 µM GrB inhibitor (24 h). When cells were pretreated with GrB inhibitor and then treated with FGF-10, the translocation of the nuclear FGFR1 was partially blocked. Immunoblotting with Lamin A/C and tubulin antibodies confirmed the specificity of the cell fractionation protocol. Nuclear FGFR1 was normalized to the nuclear marker Lamin A/C, confirming the significance of the accumulation (graph). (C) MCF-7 cells were treated with 25 µM GrB inhibitor (24 h) in serum-free medium before stimulation with FGF-10/heparin (100 ng/ml and 7.5 µg/ml, respectively). Control cells showed increased staining for nuclear FGFR1 (arrows) after FGF-10 treatment, and this was blocked by GrB inhibition. Bar, 25 µm. (D) Transwell migration assays with MCF-7 cells showed that GrB inhibition abolished the promigratory effect of FGF-10. In contrast, cells were still able to migrate in response to EGF stimulation. *, P ≤ 0.05; **, P ≤ 0.01 (Student’s t test). (E) GrB activity in lysates from serum-starved MCF-7 cells with or without FGF-10/heparin treatment (100 ng/ml and 7.5 µg/ml, respectively) in the presence or absence of pretreatment with the serine protease inhibitor 3,4-dichloroisocoumarin (DCI; 50 µM; 1-h pretreatment). Activity was measured at 405-nm absorbance at hourly intervals after the addition of 200 µM Ac-IEPD-pNA substrate. FGF-10 treatment significantly increased GrB activity in MCF-7 cells, but this effect was abrogated by pretreatment with DCI. **, P ≤ 0.01; ***, P ≤ 0.001 (Student’s t test). (F) GrB activity in MCF-7 cells growing in full serum is markedly reduced by treatment with 50 µM DCI (2 h). (G) 5-h treatment with 50 µM DCI decreased nuclear FGFR1 protein significantly. *, P ≤ 0.05 (Student’s t test). Error bars show means ± SEM. A.U., arbitrary unit; Ctr, control.