Fig. 7

FHC silencing induces EMT in H460 cells. a Real-time PCR analysis of FHC mRNAs expression were performed on total RNA extracted from H460^shRNA and H460^shFHC cells. Final results represent mean ± SD of three independent experiments. Statistical significance was evaluated by Student t-test (***, p < 0.001). H460^shRNA and H460^shFHC cells were fixed and incubated with monoclonal anti-FHC antibody (1:200) followed by incubation with the appropriate secondary antibody. Nuclei were visualized by DAPI staining. Images were collected using a Leica TCS SP2 confocal microscopy system (63×). Representative data from one of three experiments. b Cells were seeded in 10 ml of RPMI in 100 mm plates. After 24 h, 48 h, 72 h and 96 h, 500 μl of supernatant were taken and glucose and lactate concentration was measured. Final results represent mean ± SD of three independent experiments. Statistical significance was evaluated by Two-Way ANOVA (Sidak’s) (*, p < 0.05, ***, p < 0.001). c Real-time PCR analysis of E-Cadherin, Vimentin, Twist and Slug. mRNAs expression was performed on total RNA extracted from H460^shRNA and H460^shFHC cells. Final results represent mean ± SD of three independent experiments. Statistical significance was evaluated by Student t-test (*, p < 0.05). Western Blot analysis for Vimentin, ZEB1 and E-Cadherin were performed on 100 μg of total protein extract from H460^shRNA and H460^shFHC cells. γ-Tubulin was used as loading control. d H460^shRNA and H460^shFHC cells migration was assessed using a wound-healing assay. Images of wounded monolayer of H460 cells were taken at times 0 h, 24 h and 48 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 H460^shRNA as control. Statistical significance was evaluated by Two-Way ANOVA (Sidak’s) (n.s., not significant ****, p < 0.0001)