Fig. 3

FGF7/FGFR2 signalling promotes dissociation of Keap1/Nrf-2 complex, Nrf-2 expression and activity. A MCF7, MCF7 FGFR2(−), T47D and T47D FGFR2(−) cells were incubated with FGF7 (50 ng/ml) for 10–60 min. p62 serine 349 phosphorylation was evaluated by western blotting and quantified by densitometry. Quantitative data are presented as the mean ± SD (n = 3), **P < 0.01. Statistical comparisons were made using 2-tailed Student’s t-test. B MCF7, MCF7 FGFR2(−), T47D and T47D FGFR2(−) cells were incubated with FGF7 (50 ng/ml) for 24, 48 and 72 h. The quantification represents the relative Nrf-2 and Keap1 expression levels normalized to β-actin, mean ± SD (n = 3), *P < 0.05, **P < 0.01. Statistical comparisons were made using 2-tailed Student’s t-test. C The expression level of Nrf-2 was determined by western blot analysis in cytoplasmic and nuclear extracts of MCF7 and T47D cells treated with FGF7 (50 ng/ml) for 4 and 8 h. Vinculin and Lamin B1 were used as loading controls for the cytoplasmic or nuclear fractions, respectively. D Detection of Nrf-2 and Keap1 interactions in MCF7 and T47D cells treated with FGF7 (50 ng/ml) for 1 h by proximity ligation assay. Representative fluorescent microscopy images were taken (scale bar 10 μm). Quantitative analysis of PLA results was done using ImageJ software and presented as % of Keap1/Nrf-2 puncta per cell, mean ± SD (n = 3), *P < 0.05. Statistical comparisons were made using 2-tailed Student’s t-test. E MCF7 and T47D cells were incubated with FGF7 (50 ng/ml) for 1, 2 and 6 h. The mRNA expression level of NFE2L2 (gene encoding Nrf-2) was analyzed by qPCR. Data are shown as a ratio to control, mean ± SD (n = 3), **P < 0.01. Statistical comparisons were made using 2-tailed Student’s t-test. F MCF7 and T47D cells were treated with FGF7 (50 ng/ml) for 6 and 24 h, and nuclear extracts were analyzed for Nrf-2 binding capacity to antioxidant response element (ARE) by Nrf2 Transcription Factor Assay Kit. Data are shown as a ratio to control, mean ± SD (n = 3), *P < 0.05, **P < 0.01. Statistical comparisons were made using 2-tailed Student’s t-test. G MCF7 and T47D cells were incubated with FGF7 (50 ng/ml) for 24, 30 and 36 h, and mRNA levels of Nrf-2-dependent genes (HMOX1, NQO1, SOD1) were determined by qPCR. Data are shown as a ratio to control. All quantitative data are presented as the mean ± SD (n = 3), *P < 0.05, **P < 0.01. Statistical comparisons were made using 2-tailed Student’s t-test