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: Alternative splicing of FLNB exon 30 is strongly associated with basal-like breast cancer.(A) FLNB exon 30 PSI in all CCLE breast cancer cell lines. RT-PCR validation of FLNB exon 30 splicing in two Basal B cell lines (BT549 and MDAMB231) and two luminal cell lines (ZR75-1 and MCF7) is shown on the right. (B) CCLE cell lines (except hematopoietic cell lines, n = 787) were ranked by their FLNB exon 30 Percent Spliced In (PSI) values, and associated gene expression signatures were analyzed by the REVEALER program. The top associated signatures with their Information coefficient (IC), p values and False Discovery Rates (FDR) are shown.

Fig 4: FLNB isoform switching promotes the mesenchymal cell state.(A) Schematic of FLNB (Filamin B) protein domain structure, which contains an N-terminal actin-binding domain (ABD, shown in yellow), 24 filamin repeats (shown in blue) and two hinge domains (H1 and H2, shown in red). The exon 30 of FLNB encodes the first hinge domain (H1). (B) CD44-high and CD44-low HMLER cells were sorted and FLNB AS was analyzed by RT-PCR with primers flanking FLNB exon 30. The CD44-high HMLER cells were spontaneously generated. (C) The expression of FLNB was suppressed by siRNAs against FLNB 3’UTR region (FLNB-U1, U2 and UTR1,2) in HMLE cells. FLNB-U1,2 is a pool of FLNB-U1 and FLNB-U2 siRNAs. Protein levels of FN1, VIM (mesenchymal markers), CDH1 (epithelial marker) and FLNB after FLNB suppression was quantified by immunoblotting. (D) The expression of FLNB was rescued by FLNB-L (FLNB long isoform) or FLNB-ΔH1 (FLNB lacking H1 domain) after endogenous FLNB depletion by siRNA targeting the UTR of FLNB (FLNB-U1,2). Protein levels of FN1, VIM (mesenchymal markers), CDH1 (epithelial marker) and FLNB were quantified by immunoblotting. (E) Schematic of CRISPR/Cas9-mediated targeting of the FLNB intron 29 - exon 30 junction. (Left) Guide RNAs were designed to target the junction of FLNB intron 29 (in lower-case black letters) and exon 30 (in capital red letters). (Right) Examples of modifications to the junction induced by sgRNAs sgFLNB-SK2 and sgFLNB-SK4 are shown. (F) FLNB exon 30 skipping and total FLNB levels were quantified in cells expressing sgRNAs targeting the junction of FLNB intron 29 and exon 30 by RT-PCR (upper) and immunoblot (lower) (corresponding to Figure 6E). (G–I) Effect of CRISPR/Cas9-mediated modulation of the FLNB-L/FLNB-ΔH1 ratio on the expression of mesenchymal and epithelial markers as examined by qPCR (G), on the expression of CD44 as analyzed by flow cytometry (H), and on the number of mammosphere formed per 1000 cells cultured in low attachment condition (I). n = 4 for (G), n = 6 for (H), n = 3 for (I). *, p<0.05; **, p<0.01, Student’s two-tailed t-test compared to sgGFP controls.

Fig 5: DPP7 suppresses disulfidptosis in CRC cells. (A) Correlation heatmap showing the relationship between DPP7 and disulfidptosis‐related genes. Pearson correlation coefficients are indicated. (B) Western blot analysis of DPP7 protein expression in RKO and HC29 cells transduced with empty vector (EV) or DPP7‐expressing lentivirus. GAPDH was used as a loading control. (C, D) Cell viability of RKO (C) and HC29 (D) cells with or without DPP7 overexpression under glucose‐replete (Glc+) or glucose‐deprived (Glc‐) conditions for 12 h. (E, F) Flow cytometry analysis of apoptosis in RKO (E) and HC29 (F) cells with or without DPP7 overexpression under indicated conditions. (G) Non‐reducing (Non‐Re) and reducing (Re) Western blot analysis of cytoskeletal proteins (Drebrin, FLNA and FLNB) in RKO and HC29 cells with or without DPP7 overexpression under indicated conditions. Data are presented as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001. SEF, starvation effect factor.