Fig 2: EMT mediated by FLNB isoform switching is dependent on the FOXC1 transcription factor.(A) Analysis of nuclear and cytoplasmic fractions of FLNB. HMLE cells expressing Cas9 and sgRNAs targeting GFP (sgGFP, as control) or targeting the FLNB intron 29 - exon 30 junction to induce exon skipping (sgFLNB-SK2 and SK4, described in Figure 6E), were subjected to subcellular fractionation. The levels of FLNB proteins in the nuclear and cytoplasmic fractions were analyzed by immunoblotting and the skipping of exon 30 was confirmed by RT-PCR. The nuclear levels of FLNB is diminished when exon 30 is skipped. (B) Interaction between FLNB, FOXC1 and PBX1 in the nucleus as determined by immunoprecipitation followed by immunoblotting. HMLE cells expressing Cas9 and sgRNAs targeting GFP (sgGFP, as control) or targeting the FLNB intron 29 - exon 30 junction to induce exon skipping (sgFLNB-SK4, described in Figure 6E), were subjected to immunoprecipitation analysis using nuclear protein extract. The interaction between FLNB and FOXC1 in the nuclear extract was reduced largely due to the decrease in nuclear FLNB levels. (C) The depletion of FOXC1 diminishes the upregulation of mesenchymal markers (ZEB1 and FN1) mediated by FLNB exon 30 skipping (sgFLNB-SK2 and SK4, shown in red bars) as measured by qPCR. n = 6, *, p<0.05; **, p<0.01, Student’s two-tailed t-test. The knockdown efficiency for FOXC1 is shown in the upper panel. (D) The rate of mammosphere formation in HMLE cells that expressed the indicated sgRNAs and transfected with a FOXC1 siRNA pool. The number of mammospheres per 1000 cells plated was counted. Representative images (left) and quantification (right) are shown. n = 3, **, p<0.01, Student’s two-tailed t-test. (E, F) Depletion of FOXC1 diminishes the upregulation of mesenchymal markers (E) and mammosphere formation (F) mediated by QKI and RBFOX1 expression, as measured by qPCR (E) and mammosphere formation assay (F). n = 6 for (E), n = 4 for (F). *, p<0.05; **, p<0.01, Student’s two-tailed t-test. (G) A model of the mechanism underlying the QKI and RBFOX1-induced mesenchymal and stem-like cell state.

Fig 3: QKI is required for efficient BIN1 pre-mRNA processing. (A) Representative western blot (top) of protein extracted from three independent replicates of HuH7 WT or HuH7 QKI-KO cells co-transfected with pDUP-BIN1 exon 7 splicing reporter and either vector control (vec), myc:Qk5 (wtQk5) or K120A/R124A myc:Qk5 (mtQk5) probed with panQKI antibody (green) and Tubulin antibody (magenta). Representative RT-PCR and BioAnalyzer gel-like (bottom) image showing splicing reporter products derived from RNA extracted from WT or QKI-KO cells described above. (B) Bar graph showing mean percentage of each RT-PCR product (or products) observed in (A), under each experimental condition described (error bars denote standard deviation from the mean; ns denotes not significant, *P < 0.05, **P < 0.01; note that in the absence of minus reverse transcription controls we cannot exclude the possibility that reporter DNA might have been amplified and co-migrate with ‘unspliced’). (C) Overview graphic of pDUP-BIN1 exon 7 splicing reporter (top) with WT and mutant sequences shown in inset below. Representative RT-PCR and BioAnalyzer gel-like image (bottom) showing quantification of WT or mutant splicing reporter products derived from RNA extracts from WT HuH7 cells transfected in triplicate; the mean percentage observed of each product(s) is shown below the gel-like image represented by intensity plot (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by Student's t test; see also Supplementary Figure S6B for mean values ± standard deviation and minus reverse transcription controls). (D) RT-qPCR of RNA extracted from d8 c.myo NKX2.5?EGFP WT, polyKO, or 4–2KO cells measuring (from left to right) total exon 6 (‘total’; primers internal to exon 6 including common exon 6 forward), intron 6 included (‘int6+’; common forward primer in exon 6 with reverse primer in intron 6), exon 6–7 spliced (‘ex7+’; common forward primer in exon 6 with reverse primer spanning exon 6:7 junction), exon 6–8 spliced (‘ex7–’; common forward primer in exon 6 with reverse primer spanning exon 6:8 junction), exon 6–8 spliced (‘ex7-’; forward primer spanning exon 6:8 junction with common reverse primer in exon 8) exon 7–8 spliced (‘ex7+’; forward primer spanning exon 7:8 junction with common reverse primer in exon 8), intron 7 included (int7+; forward primer in intron 7 with the common reverse primer in exon 8), and total exon 8 levels (‘total’; forward primer in exon 8 with the common reverse primer in exon 8) from the endogenous BIN1 transcript; each value is shown normalized to HMBS mRNA and relative to WT; graphic below bars indicates approximate location of forward and reverse primer pairs; n = 3 biological replicates, ns = not significant, *P < 0.05, **P < 0.01, ***P < 0.001, relative to WT by Student's t-test). (E) RT-qPCR of RNA extracted from d8 c.myo NKX2.5?EGFP WT or polyKO cells’ cytoplasmic (cyto), nucleoplasmic (nucl), or chromatin (chr) fractions, measuring XIST (top) or 7SK (bottom) RNAs. Their levels are shown normalized to HMBS mRNA and relative to total (unfractionated) cell extract RNA levels (**P < 0.01, ***P < 0.001, ****P < 0.0001 by Student's t test). (F) Western blot of protein extracted from subcellular fractions of WT (left) or polyKO (right) d8 c.myo NKX2.5?EGFP cells as described above and probed with anti-GAPDH (magenta) and anti-histone H3 (green) antibodies. (G) RT-qPCR of RNA extracted from subcellular fractions of WT or polyKO d8 c.myo NKX2.5?EGFP cells as described above and measuring HMBS (left) or BIN1 (right) RNA, shown as the percentage of RNA observed by compartment, relative to total RNA from unfractionated extracts; ns denotes not significant, *P < 0.05, **P < 0.01, ***P < 0.001 by Student's t test.

Fig 4: Qki5 and Qki6 are expressed in early neural stem cells of the developing mouse brain. (A) Frontal sections of mouse brains at each developmental age (embryonic day 9.5 [E9.5], E12.5, E15.5, and E18.5) were immunostained using anti-pan-Qki antibodies or isoform-specific antibodies (Qki5, Qki6, and Qki7). This staining revealed that Qki5 and Qki6 are expressed in the ventricular zone (VZ) at E9.5, when neural tube closure is complete. Expression of these proteins continues through embryogenesis. Expression of Qki5 and Qki6 in neural stem cells is down-regulated during neurogenesis. Bar, 100 µm. (B) Detailed expression of Qki5 and lineage marker proteins in an E15.5 mouse cortex. Qki5-positive cells (green) also express Qki6 (red) in the VZ, although the subcellular localization of these proteins is quite different, with Qki5 being present in the nucleus, and Qki6 present in the cytoplasm. Qki5 expression colocalizes with Pax6 (red), a neural stem cell marker, and is down-regulated in Tbr2-positive intermediate progenitors (red) in the VZ and subventricular zone (SVZ). Qki5 and NeuroD (red) are mutually exclusive, and Qki5 expression is completely absent in Tag1-positive axons (red). Bar, 100 µm. (C) Mutually exclusive subcellular localization of Qki5 and Qki6 proteins in embryonic neural stem cells from an E15.5 mouse neocortex. Qki5 (red) is clearly distributed in the nucleus stained with Hoechst 33258 (blue). In contrast, Qki6 (green) is localized mainly in the cytoplasm. The bottom two panels are higher-magnification views of the boxed area of the top panels. Bars: top panels, 20 µm; higher-magnification views, 10 µm. (D) Schematic illustrations of the expression patterns of Qki proteins in the developing neocortex. (Left panel) Qki5 and Qki6 are expressed in embryonic neural stem cells at E9.5, and Qki7 expression starts at around E18.5. (Right panel) The expression of Qki proteins with lineage marker proteins in the E15.5 mouse neocortex. Qki5/6 expression is notably down-regulated in Tbr2+ intermediate progenitors. (CP) cortical plate; (IZ) intermediate zone.

Fig 5: Endogenous QKI and RBFOX1 isoform expression in HME and HMLER cells.(A) Expression of endogenous QKI isoforms based on RSEM quantification of RNA-sequencing data in HME cells expressing control EGFP, RBFOX1, or SNAI1. (B) RT-PCR for alternatively spliced exons in endogenous RBFOX1 in HME cells expressing control EGFP, SNAI1 or QKI. (C) Immunoblots for RBFOX1 protein expression in HME or HMLER cells.