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. 2018 Apr 4;8(4):170274. doi: 10.1098/rsob.170274

Figure 1.

Figure 1.

Ectopic expression of SCP4 enhances TGFβ-induced EMT and promotes migration in MCF10A. (a) SCP4 promotes TGFβ-induced morphological changes. MCF10A cells stably expressing SCP4 or GFP were treated with TGFβ (1 ng ml−1) for 48 h. To block TGFβ signalling, 5 µM SB431542 was added 1 h before TGFβ stimulation. Scale bars, 100 µm. (b) SCP4 accelerates TGFβ-induced migration. Confluent monolayers of MCF10A cells stably expressing SCP4 or GFP were scratched with a pipette tip to create a cell-free line. The wounded cell monolayers were incubated in OPTI-MEM medium with 1 ng ml−1 of TGFβ alone or in combination with inhibitor SB431542 for the indicated time periods. The wound healing was recorded by microphotography of the same region. Scale bars, 200 µm. (c) SCP4 enhances cell invasiveness. MDA-MD-231 cells stably expressing SCP4 or GFP were cultured in the transwell culture chambers with an 8 µm pore-size polycarbonate filter according to the manufacturer's instructions. Eight hours later, invaded cells that were attached to the lower surface of the filter were stained with DAPI. The slices were imaged. Scale bars, 200 µm. (d) SCP4 increases TGFβ-induced EMT marker expression. MCF10A cells stably expressing SCP4 or GFP were treated with 1 ng ml−1 TGFβ alone or in combination with SB431542 for 48 h. Levels of E-cadherin and Snail were analysed by western blotting. (e) SCP4 enhances TGFβ-induced E-cadherin mRNA inhibition. MCF10A cells stably expressing SCP4 or GFP were treated with 1 ng ml−1 TGFβ alone or in combination with SB431542 for 48 h. Total mRNA was extracted for RT–PCR analysis. Data are shown as mean ± s.e.m. (triplicate assays). *p < 0.05 SCP4 versus GFP. (f) SCP4 enhances TGFβ-induced fibronectin mRNA expression. Experiment was carried out as described for (e). Data are shown as mean ± s.e.m. (triplicate assays). *p < 0.05 SCP4 versus GFP. (g) SCP4 enhances TGFβ-induced vimentin mRNA expression. Experiment was carried out as described for (e). Data are shown as mean ± s.e.m. (triplicate assays). *p < 0.05 SCP4 versus GFP.