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. 2016 Feb 18;7(13):15811–15827. doi: 10.18632/oncotarget.7515

Figure 6. Overexpression of ARF controls motility, invasion, proliferation, and survival of MCF7 cells.

Figure 6

A. MCF7 were transfected with empty vector (cnt), or ARF1 or ARF6. After 48h, confluent cells were scratch and wound healing was monitored over 16h in the presence FBS. Hoechst-stained nuclei were tracked using Fiji software. Representative pictures of cells tracking over 16h are shown. Scale bars, 50 μm. Quantification of cell motility: right graphs represent the mean total and net displacement. n = 120-150 cells. **p < 0.001,****p < 0.0005. B. Cells transfected as in A, were seeded into Matrigel-coated Boyden chambers. Cells were left untreated or stimulated with FBS 10% for 16 hours. Graph is representative of three images taken, per condition, of three independent experiments. C. Protein expression of pro-MMP2, HA-tagged protein and actin were detected by Western immunoblotting. D. Transfected cells were stimulated or not for 12 hours with 10% FBS. Supernatants were collected and analyzed by zymography. Data are the mean ± SEM of three experiments. E. Cells were transfected with either empty vector, HA-ARF1, scrambled or two different FAK siRNA. Protein expression of pro-MMP2, FAK, HA-tagged protein and actin were detected by Western immunoblotting. Graph represents the mean ± SEM of five experiments. Significance was measured by one-way ANOVA followed by Bonferroni's multiple comparison tests. *p < 0.05, **p < 0.01. F. MCF7 cells transfected as in A, were seeded in 96-well plates. Proliferation was measured by MTT (Thiazolyl blue-tetrazolium-bromide) over 7 days. These experiments are representative of four performed in triplicate. G. Endogenous expression of Ki-67, ARF6 and actin was detected by Western immunoblotting, after 5 and 4 days. H. Cells were transfected as in A. and 24 hours after the transfection, they were treated with vehicle or etoposide. After 48 hours, surviving cells were analyzed by FACS, using Annexin-V binding and PI permeability assays. Data are the mean ± SEM of three experiments respectively. I. Transfected cells treated as in H. and level of pro-caspase 9, pro-caspase 3, actin and HA-tagged protein were detected by Western immunoblotting. Data are the mean ± SEM of three experiments. In D., H. and I., significance was assessed by two-way ANOVA followed by Bonferroni's multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001.