Fig. 3: Functional characterization of SF3B1 missense variants in K562 cells.
From: De novo variants in the splicing factor gene SF3B1 are associated with neurodevelopmental disorders
A RT-PCR analysis of exon skipping of DUSP11 and RBM5, used as indicators of SF3B1 loss-of-function, in K562 cells co-expressing siSF3B1 and various NDD-associated variants. The splicing deficient SF3B1 “ins” isoform32 was used as a control. B Digital quantification of exon skipping in DUSP11 and RBM5 (n = 3). C Steady-state SF3B1 protein levels detected by Western-Blot in K562 cells stably expressing four NDD variants under the control of a doxycycline-inducible promoter, in combination with endogenous SF3B1 silencing (shSF3B1). Total SF3B1 proteins (endogenous and recombinant) were detected using anti-SF3B1 antibody. Recombinant SF3B1 was detected using anti-FLAG antibody. D Proliferation curves of inducible K562 cells (as in D) following induction by doxycycline (2 microg/mL) (n = 4 biological replicates for all conditions, except for shSF3B1 alone for which n = 2). A two-sided Mann–Whitney test was applied and showed a significant difference (p value < 0,05) between K562 cells expressing WT, N829S, E722K or P780L variants and K562 cells expressing E980* variant (p value = 0,0286) or the inactive SF3B1ins splicing isoform (p value = 0,0286). Data are presented as mean values ± SD. E RT-PCR detection of aberrant transcripts known to be specifically produced upon expression of somatic SF3B1 mutations (K700E), in K562 cells transiently expressing SF3B1 variants of interest. This experiment was repeated three times independently with similar results. In (B and D), n refers to biological replicates.
