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. 2020 Jun 3;123(4):633–643. doi: 10.1038/s41416-020-0873-z

Fig. 2. Knockdown of SLFN5 induces EMT in epithelial BRCA cell lines and promotes invasion in vitro and in vivo.

Fig. 2

a Contrast-phase and fluorescent images of MCF7 cell lines stably transfected with lentiviral control shRNA or lentiviral SLFN5 shRNA. Scale bar: 20 μm. b WB analysis of the expression levels of SLFN5, epithelial markers (E-cad and Zonula occludens protein-1 (ZO-1)) and mesenchymal markers (vimentin and N-cadherin (N-cad)) in MCF7 and T47D cell lines stably transfected with control shRNA or SLFN5 shRNA. Protein band intensity is quantified and presented as the mean ± SD of three independent experiments. **P < 0.01. c Real-time PCR analysis of gene expression in MCF7 and T47D cell lines stably transfected with control shRNA or SLFN5 shRNA. d Traditional PCR followed by agarose gel electrophoresis was performed to identify the size of amplified fragments. e–g Immunofluorescence analyses of SLFN5, E-cadherin and vimentin expression in stably transfected control shRNA or SLFN5 shRNA MCF7 cell lines, stained with the indicated primary antibodies and Alexa Fluor 594-conjugated secondary antibodies. The results were observed with laser confocal fluorescence microscopy. DAPI staining revealed the nuclei. h Migration and invasion assays of MCF7 and T47D cell lines stably transfected with control shRNA or SLFN5 shRNA. Migration and invasion cells per field were calculated. i Chick CAM invasion analysis of MCF7 cell lines stably transfected with control shRNA or SLFN5 shRNA. The white dotted lines indicate the position of the CAM. The figures shown are representative results of three similar experiments. The white arrowheads indicate cells invading into the chick embryos. The double-headed arrow indicates the depth of cell invasion. DAPI staining of the cell nuclei of both human cancer and chick cells is shown. Cell numbers and depth were quantified as the mean ± SD of three independent experiments. j Rescue experiments were performed using human SLFN5 viral particles (because mouse Slfn5 has only 59% homology to humans). The results show that SLFN5 transfection partially rescued SLFN5 knockdown-induced E-cadherin and vimentin changes. Protein band intensity is quantified and is presented as the mean ± SD of three independent experiments. **P < 0.01.