Fig. 2: Validation of the reporter in cell culture.

A Transfection of the reporter in HEK293 cells – upper panel: transient transfections (24 h) show both dsRED and EGFP signal due to multiple copies of the reporter; lower panel – stable transfected cells display only dsRED fluorescence corresponding to the VEGF-A terminal exon proximal splice site usage in these cells. B RT-PCR for the reporter shows correct splicing in HEK293 cells. C FACS analysis of HEK293 stably transfected with the reporter shows dsRED fluorescence only (parentFluor area is the trace for naïve untransfected cells). D In proliferating conditionally immortalised human podocytes, selection of the reporter’s distal splice site leads to EGFP expression. No dsRED, only EGFP expression was observed in transfected podocytes indicating an anti-angiogenic splicing pattern (upper panel). In Denys Drash Syndrome (DDS) podocytes, where the VEGF-A_xxxb isoforms are not expressed, we see only dsRED expression. Transfection efficiency is estimated at 20%. E SRPK1 inhibition increases use of the pRG-VEGF8ab reporter distal splice site (dsRED/EGFP), as measured by the plate reader. n = 3, *P < 0.05. No significant difference in dsRED+EGFP measurements. F The reporter was further validated after stable transfection into PC3 cells, Both SRPK1 inhibitors resulted in an increase in the EGFP/dsRED ratio. n = 3, *P < 0.05 vs DMSO. G To determine whether the reporter splicing mimics that of endogenous VEGF-A, PC3 cells were treated with the same SRPK1 inhibitors. The protein VEGF-A₁₆₅b/panVEGF-A₁₆₅ ratio was increased after treatment with SRPIN340 and SPHINX, although only significant with the more potent SRPK1 inhibitor, SPHINX. n = 3, *P < 0.05 vs DMSO.