FIGURE 5.

SPF45/SR140/CHERP regulate FOXM1 AS. (A) Scheme of AS events involving FOXM1 exon 9 (upper panel) and their changes upon knockdown of SPF45, SR140, or CHERP measured by RT-PCR followed by capillary electrophoresis (lower panel). The isoform identity of the amplification products is indicated. (B) Validation of FOXM1 exon 9 AS events (solid bars: full exon inclusion; striped bars: shorter exon inclusion) changes measured as in A in HeLa and U2OS cells. Data are represented as mean ± SD of three biological replicates, and P-values are calculated comparing to shCNT by Student's t-test ([*] P < 0.05; [**] P < 0.01; [***] P < 0.001). (C) Structural domain organization of FOXM1 isoforms. The different domains are indicated as well as the location and likely functional impact of inclusion of sequences corresponding to exon 6 and exon 9 inclusion. PTC: premature termination codon. (D) Fold change over shCNT of mRNA levels of FOXM1 isoforms and total FOXM1 quantified by RT-qPCR upon knockdown of SPF45, SR140, or CHERP in HeLa and U2OS cells. Data are represented as mean ± SD of three biological replicates, and P-values are calculated by comparing to shCNT by one-way ANOVA followed by Dunnett's multiple comparison test ([*] P < 0.05; [**] P < 0.01; [***] P < 0.001). (E) Relative abundance of the indicated common or isoform-specific peptides in control versus SPF45-depleted cells by targeted proteomics, normalized by the levels of ACLY, and corresponding fold changes (in log₂) between the two conditions. (F) Relative expression levels of known targets of transcriptional activation by FOXM1, quantified by Cuffdiff in RNA-seq data sets of knockdown of SPF45, SR140, or CHERP in HeLa cells. Gene expression is displayed as FPKM values (y-axis) and significant values—according to Cuffdiff differential gene expression test—are indicated by an asterisk. See also Supplemental Figure S7.