Figure 6. QKI expression levels influence pre-mRNA splicing during THP-1-based monocyte-like to macrophage-like cell differentiation.

(a) Schematic depicting detectable alternative splicing events with the splicing-sensitive microarray platform and number of inclusion (incl.; top lines) or exclusion (excl.; bottom lines) events observed in unstimulated THP-1 ‘monocytes' (left) and 3-day PMA-stimulated THP-1 ‘macrophages' (n=3, q≤0.05). (b) Scatterplots of skip (y axis) and include (x axis) probe set intensity for selected alternative splicing events in sh-Cont (blue boxes) versus sh-QKI (orange circles) in unstimulated and 3 days PMA-stimulated THP-1 ‘monocytes' and ‘macrophages', respectively. Regression coefficients (constrained to pass the origin) are depicted as solid lines. The log₂ difference in the slopes (termed separation score; ss) are provided to the right of the plots for each event, with for example, an ss of −1.72, indicating a 3.3-fold more inclusion of ADD3 exon 13 in sh-QKI versus sh-Cont THP-1 ‘monocytes'. Full event details are provided in Supplementary Data 6. CE, cassette exon; Alt 5′ or 3′, alternative 5′ or 3′ splice site; RI, retained intron. (c) SpliceTrap assessment of average proximal ACUAA RNA motif enrichment in 50 bp windows upstream and downstream of alternatively spliced cassette exons as compared with a background set of exons (grey circles). The relationship between the frequency of exon exclusion (blue triangles) or exon inclusion (red squares) and ACUAA RNA motif enrichment are depicted. (d) PCR validation of alternatively spliced cassette exons in sh-Cont and sh-QKI THP-1 ‘monocytes' and ‘macrophages'. Primers were designed to target constitutive flanking exons. PCR product size for exon inclusion (top) and exclusion (bottom) variants are provided (left). All experiments depict biological n=3. (e) PCR validation of three splicing events in wt and qk^v mouse-derived primary monocytes and 7 days M-CSF-stimulated macrophages. PCR product size for exon inclusion (top) and exclusion (bottom) variants are provided (left). Depicted is a representative PCR for at least a biological n≥3.