Fig. 5
From: Epithelial-to-mesenchymal transition in FHC-silenced cells: the role of CXCR4/CXCL12 axis
ROS levels and EMT in MCF-7shFHC cells. a MCF-7shRNA and MCF-7shFHC cells (106) were incubated for 15 min with 20 μM of 2′-7′-DCF and washed with HBSS solution. Fluorescence was measured using the FACS FORTESSA. Final results represent mean ± SD of three independent experiments. Statistical significance was evaluated by Student t-test (*, p < 0.05). b Western Blot analysis for E-Cadherin and Vimentin expression was performed on 50 μg of total protein extract from MCF-7shRNA and MCF-7shFHC cells treated with NAC (2,5–5-10 mM). γ-Tubulin was used as loading control. Representative data from one of three experiments. c Real-time PCR analysis of E-Cadherin, Vimentin, Twist and Slug mRNAs expression were performed on total RNA extracted from MCF-7shRNA, MCF-7shFHC and MCF-7shFHC cells treated with 5 mM NAC for 2 h. Final results represent mean ± SD of three independent experiments. Statistical significance was evaluated by Student t-test (*, p < 0.05, ****, p < 0.0001, n.s., not significant). d Wound healing assay was conducted to measure migration capacity of MCF-7shRNA and MCF-7shFHC cells treated and not treated with NAC (5 mM). Images of cellular migration were taken at times 0 h and 24 h (magnification of 10X) using The Leica DFC420 C and Leica Application Suite Software. Wound size was quantified by ImageJ 64 software. Final results represent mean ± SD of three independent experiments. Cell migration fold change was evaluated using the T0 of MCF-7shRNA as control. Statistical significance was evaluated by Two-Way ANOVA (Sidak’s) (n.s., not significant). e Proliferation rate of MCF-7shRNA and MCF-7shFHC cells with NAC (10 mM) was assessed by cell count at 24, 48 and 72 h. Final results represent mean ± SD of three independent experiments each performed in triplicate. Statistical significance was evaluated by Two-Way ANOVA (Sidak’s) (**, p < 0.01). f MCF-7shRNA and MCF-7shFHC cells treated with NAC 5 mM were fixed and incubated with Oregon Green 488 phalloidin (1:400) to visualize actin filaments. Nuclei were visualized by DAPI staining. Images were collected using a Leica TCS SP2 confocal microscopy system (40X). Representative data from one of three experiments