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Fig. 6 | Journal of Experimental & Clinical Cancer Research

Fig. 6

From: Epithelial-to-mesenchymal transition in FHC-silenced cells: the role of CXCR4/CXCL12 axis

Fig. 6

CXCR4 axis is activated in MCF-7shFHC cells. a Western Blot analysis for CXCR4 expression was performed on 50 μg of total protein extract from MCF-7shRNA and MCF-7shFHC cells. γ-Tubulin was used as loading control. Representative data from one of three experiments. The graph represents the mean of the optical densities. Statistical significance was evaluated by Student t-test (*, p < 0.05). b Cells were stained with anti-CXCR4 PE (10 μL/1 × 106 cells) and evaluated by a FACS Canto II cytofluorometer. Final results represent mean ± SD of three independent experiments. Statistical significance was evaluated by Student t-test (n.s., not significant). c cAMP production was verified after Forskolin (1 μM), CXCL12 (100 ng/mL), and AMD3100 (10 μM) treatment in MCF7 cells. Final results represent mean ± SD of three independent experiments. Statistical significance was evaluated by Two-Way ANOVA (Tukey’s) (n.s., not significant). d Western Blot analysis for pERK1/2 was performed on 50 μg of total protein extract from MCF-7shRNA and MCF-7shFHC cells. Cells were stimulated with CXCL12 (100 ng/ml) at the indicated time points. ERK1/2 was used as loading control. Representative data from one of three experiments. Numbers below the western blot indicates sample progression. e Upper: Western Blot analysis for pAKT was performed on 50 μg of total protein extract from MCF-7shRNA and MCF-7shFHC cells. Cells were stimulated with CXCL12 (100 ng/ml) at the indicated time points. AKT was used as loading control. Numbers below the western blot indicates sample progression. Lower: Cells were stimulated with CXCL12 (100 ng/ml for 10 min) and treated with the AMD3100 (10 μM) one hour before their exposure to CXCL12. AKT was used as loading control. Numbers below the western blot indicates sample progression. Representative data from one of three experiments. f Western Blot analysis for pP70S6 K expression was performed on 50 μg of total protein extract from MCF-7shRNA and MCF-7shFHC cells. Cells were stimulated with CXCL12 (100 ng/ml for 10 min) and treated with the AMD3100 (10 μM) one hour before their exposure to CXCL12. P70S6 K was used as loading control. Numbers below the western blot indicates sample progression. Representative data from one of three experiments. g CXCL12 dependent-cell migration was examined in 24-well plates. Cells were placed in the upper chamber (8 μm) in the presence of AMD3100 (10 μM). Cells migrated toward CXCL12 (100 ng/ml) for 18 h. The cells were counted in ten different consecutive high power fields (magnification 200×). Final results represent mean ± SD of three independent experiments. Statistical significance was evaluated by Two-Way ANOVA (Tukey’s) (*, p < 0.05, ***, p < 0.001, ****, P < 0.0001).

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