Fig 2: SLU7 controls SRSF1 and SRSF3 pre-mRNAs splicing and prevents the induction of SRSF3-TR forms. (A) Schematic representation of SRSF1 and SRSF3 genes and the different mRNAs generated by alternative splicing. The location of primers used for PCRs are indicated. (B) PCR detection of SRSF3 and SRSF1 isoforms 48 h after transfection with siGL or siSLU7 in the indicated cell lines. The aberrant isoforms are indicated by arrowheads. (C) Western blot analysis of SRSF1, SRSF3 (antibody from MBL), ?-H2AX and SLU7 in PLC/PRF/5, HeLa and H358 cells 48 h after transfection with siGL and siSLU7. Actin was used as loading control. (D) Western blot analysis of truncated SRSF3 forms (antibody from Thermo) in H358 cells 48 h after transfection with siGL, siSLU7 or siSLU7+siISO2. Western blot analysis of SLU7 and Actin protein levels as loading control are shown. Upper right panels show different exposures of the same membrane probed with anti-SRSF3 antibody to better identify the truncated SRSF3 forms. Lower right panels show the RT-PCR analysis of SRSF3-ISO2 and SLU7 mRNAs. All experiments were performed at least three times with biological duplicates per condition.

Fig 3: SRSF1/SRSF3 knockdown and SRPK1 inhibitors abrogate HUVEC-RFP invasion and sprouting in 3D collagen matrix. a Schematic representation of 3 different types of endothelial cell HUVEC-RFP behavior. #1: aborted endothelial cell sprouting, endothelial cells invade the collagen matrix without being followed by stalk cells; #2: capillary formation, complete process of endothelial cell invasion and sprouting. Filopodia projecting from the tip cells are observed; #3 early step of endothelial cell responses. For quantification, cell responses were scored according to these 3 types of cell behavior. b HUVEC-RFP were transfected for 48 h with control siRNA (mismatch) or a 50:50 mixture of two different SRSF1 (SRSF1) or SRSF3 (SRSF3) siRNAs before being analyzed in 3D invasion assay. Upper panel: immunoblots showing the efficient knockdown of SRSF1 or SRSF3 in HUVEC-RFP. GAPDH was used as a loading control. Lower panels: mosaic of confocal images covering around 300 µm of matrices border for different HUVEC-RFP post-transfection with indicated siRNA. HUVEC-RFP cells are in red and nuclei in blue. Scale bar = 100µm. Bar charts represent the quantifications of the invading distance of HUVEC-RFP within the 3D collagen matrices and the number of invading cells (nuclei) per 300 µm of gel border. Each black dot indicates one invading sprout. c HUVEC-RFP cells were treated or not (NT) with 3nM FGF-2 for 24 h in the presence or absence of 5µM SPHINX31 or 10µM SRPIN340 before performing 3D invasion assay. Left top panels: transmission light microscopy images showing invasive capacities of HUVEC-RFP within the 3D collagen gels. Left bottom panels: mosaic of confocal images covering around 300 µm of matrices border. HUVEC-RFP cells are in red and nuclei in blue. Scale bar = 100µm. Right panel: Bar charts represent the quantification of the invading distance of HUVEC- RFP within the 3D collagen matrices (top) and the number of invading cells (nuclei) per 300 µm of gel border (bottom). Each black dot indicates one invading sprout. b, c Graphs represent mean values ± SD of three independent experiments. Unpaired t test, ***p<0.001

Fig 4: FGF-2 increases SRSF1, SRSF3 and SRPK1 protein levels in primary endothelial cells. a, b xCELLigence (a) or MTS (b) assay was used to assess HUVEC and HDMEC cellular adhesion, proliferation, and viability in response to FGF-2 at 1 or 3nM. Data are representative of at least 2 independent experiments performed in at least triplicate (mean ± SD, unpaired t test, **p<0.01; ***p<0.001). c Representative immunoblots for SRSF1, SRSF3, SRPK1 (left panel), and phospho-SR (p-SR) (mAb104, right panel, two exposure times) protein levels in HUVEC and HDMEC, respectively, treated with 1 or 3nM FGF-2 for 72 h. GAPDH was used as a loading control. NT: nontreated. d Semi-quantification using ImageJ software of SRSF1, SRSF3, P-SRSF3, or SRPK1 signal relative to GAPDH signal. Ratio obtained for NT group was arbitrarily assigned the value 1. Numbers below the graph indicate the number of biological replicates for each condition (mean ± SD, unpaired t test, *p<0.05, **p<0.01). e HDMEC were treated with 1 or 3nM FGF-2 as in (c). SRSF1, SRSF3, and SRPK1 mRNA levels were quantified by RT-qPCR in each condition. GAPDH was used as an internal control. Mean ± SEM are presented (n=4, unpaired t test, **p< 0.01, ***p<0.001, ns: not significant)

Fig 5: miR-17 rescues cell-cycle arrest and prevents the induction of SRSF3-TR proteins upon SLU7 knockdown. (A and B) Cell-cycle arrest A and loss of SCC detected by chromosome spreads B (scale bar: 10 µm) induced in PLC/PRF/5 cells 48 h after SLU7 knockdown was prevented by co-transfection with miR-17. (C) The aberrant splicing of sororin induced in PLC/PRF/5, HeLa and H358 cells 48 h after SLU7 knockdown was also prevented by co-transfection with miR-17. (D) Co-transfection of miR-17 with siSLU7 recovered sororin protein levels downregulated by siSLU7 in PLC/PRF/5 cells. (E) SRSF3 exon 4 was cloned as 3'UTR in the pMIR-REPORT luciferase plasmid (pEXON4). The same sequence with the mutations at the miR-17 recognition motif marked in bold was also cloned (pMut) as control. PLC/PRF/5 and HeLa cells were transfected with pEXON4 or pMut plasmids and miR-17 or a control miRNA (Mock). Luciferase activity was analyzed 48 h after transfection. *P < 0.05. (F) The expression of SRSF3-ISO2 was measured by real time PCR in PLC/PRF/5, HeLa and H358 cells 48 h after transfection with siGL, siSLU7 or siSLU7 + miR-17. *P < 0.05, **P < 0.01. (G) Western blot analysis of SRSF3 in H358 cells 48 h after transfection with siGL, siSLU7 or siSLU7 + miR-17. The truncated forms of SRSF3 are indicated by arrows. (H) The generation of aberrant splice variants of SRSF1 induced upon SLU7 knockdown (siSLU7) in PLC/PRF/5 and HeLa cells is prevented by co-transfection with miR17. The expression of SRSF3-ISO2 and SLU7 is shown as control. (I) The induction of R-loops after SLU7 knockdown was significantly reduced after co-transfection with miR-17 in PLC/PRF/5 cells (R-loops were detected by immunofluorescence with the S9.6 antibody). Scale bar: 10 µm. The right panel shows the quantification of RNA–DNA hybrids per nucleus with ImageJ software. The DAPI signal was used to create a mask of the nucleus. The nuclear S9.6 signal intensity was then determined. The number of nucleus analyzed per condition is indicated. The plot was represented using GraphPad Prism. Red bars represent the median. ****P < 0.0001 (Mann–Whitney U-test). All experiments were performed at least three times with biological duplicates per condition.