Fig. 3

SRSF1 interactions with the fibronectin EDA exon is regulated by SNAIL1. A SRSF1 protein amount in Snai1 KO MEFs. Control and Snai1 KO MEFs were lysed in SDS buffer after the indicated times of TGFβ₁ treatment (5 ng/mL), and protein levels were analyzed by Western blotting. B SNAIL1 and SRSF1 colocalize in the nucleus of MEFs. Control and Snai1 KO MEFs were grown on glass coverslips, treated with TGFβ₁ for 24 h and fixed with 4% PFA. The cellular distributions of SNAIL1 and SRSF1 were analyzed by immunofluorescence with specific antibodies. Images were obtained by confocal microscopy. Phalloidin (pink) and DAPI (blue) staining corresponding to depicted cells are shown into a box. Merge images in control MEFs were produced with ImageJ, and colocalization is shown in yellow. C The SNAIL1 and SRSF1 interaction is RNA dependent. Extracts of MEFs treated with TGFβ₁ for 3 h were obtained in RIPA buffer, and half of the sample was treated with 400 µg/mL RNase A. RT-qPCR for total fibronectin confirmed the complete elimination of RNA in the samples. Immunoprecipitation was performed using an antibody specific for SRSF1 and agarose beads. Immunoprecipitated proteins were analyzed by Western blotting. D SNAIL1 does not bind to the fibronectin exon 33 RNA. RNA immunoprecipitation (RIP) was performed using an antibody specific for SNAIL1 or an unspecific IgG in samples of MEFs transfected to overexpress Snai1-HA (Additional file 1: Fig. S3) and treated with 5 ng/mL TGFβ₁ for 3 h. RNA enrichment in the immunoprecipitates was analyzed by RT-qPCR using primers for exon 33. Bars show binding enrichment as compared to immunoprecipitation using IgG. E SRSF1 binds to the fibronectin exon 33 RNA in a SNAIL1-dependent manner. RIP was performed using an antibody specific for SRSF1 in samples of MEFs transfected to overexpress Snai1-HA (Additional file 1: Fig. S3), or of MEFs KO for Snai1 treated with 5 ng/mL TGFβ₁ for 3 h. RNA enrichment in the immunoprecipitates was analyzed by RT-qPCR using primers for exon 33 or HPRT (as a control). Bars show binding enrichment compared to immunoprecipitation using unspecific control IgG. At least three replicates were performed for each immunoprecipitation. F and G SRSF1 and SNAIL1 bind to the EDA coding region in a TGFβ-dependent manner. ChIP was performed with an antibody specific for SRSF1 (F) or SNAIL1 (G) in samples of MEFs transfected to overexpress SNAIL-HA that were treated or not with TGFβ₁ for 3 h. Precipitated DNA was analyzed by qPCR using primers targeting Fn1 promoter (+ 116/ + 265), Fn1 exon 7 and Fn1 exon 33 (EDA). Bars show binding enrichment as compared to immunoprecipitation using unspecific IgG. At least three replicates were performed for each immunoprecipitation