Fig 2: Identification of Cooperating TSGs in an In vivo Loss-of-Function CRISPR-Cas9 Screen(A) Experimental schema for CRISPR-Cas9 library in vivo screen for cooperating TSGs. The library was packaged in lentivirus used to infect MCF10F cells at an MOI = 3. Cells were selected for expression of the library and implanted into NOD-SCID mammary fat pads at 1 × 106 cells per gland. Tumors were excised and sequenced for analyses.(B) H&E staining of tumors for identification of histological phenotypes: squamous (i and v), metaplastic (ii), papillary (iii and vi), and adenocarcinoma (iv).(C) Immunohistochemistry (IHC) staining of tumors for identification of specific epithelial and proliferation markers: EpCAM (i), Ki67 (ii), PR(203) (iii), PR(403) (iv), E-cadherin (v), p53 (vi). Scale bars represent 100 μm. Magnification = 203 for microscope images.(D) Pie charts representing the clonal heterogeneity and dominant contributing and/or cooperating TSGs in the tumors. Each chart represents one tumor; each slice of each pie chart represents a subclone.(E) Mutual exclusivity analysis of FUBP1 (gene A) and tumor suppressor genes that cooperated with FUBP1 in the screen (gene B) across 69,310 human cancer samples in 233 studies using cBioPortal. “Neither” represents the number of samples in which neither gene A nor B was altered. “A” represents the number of samples in which only gene A is altered. “B” represents the number of samples in which only gene B was altered. “A&B” represents the number of samples in which both genes A and B were altered.

Fig 3: Exons Upstream of FUBP1-Regulated Splice Sites Exhibit Diminished m6A Levels in FUBP1-Null Cells(A) Western blot validation of significant proteins from IP/MS experiment.(B and C) Dot blot measuring global m6A levels in mRNA of indicated cell lines (B), quantified in (C).(D) RNA-IP with m6A-modified or non-modified RNA bait followed by immunoblotting for a bona fide m6A reader, YTHDF2, and FUBP1.(E) Distribution of m6A-seq peaks across the CASP8, BRCA1, and MAGI3 loci, based on analysis of previously published m6A-seq data in HepG2 cells. The locations of the putative m6A sites are indicated within exons directly upstream of splice sites yielding AS transcripts found in FUBP1-null cells.(F) Relative m6A levels at m6A consensus sites of CASP8, BRCA1, and MAGI3 in exons upstream of splice sites that yield alternative variants, determined by m6A RIP-qPCR in NTC and FUBP1-null MCF10F cells.(G) CASP8, BRCA1, and MAGI3 mRNA levels relative to GAPDH determined by quantitative real-time PCR in NTC and FUBP1-null MCF10F cells (n = 2) using primers flanking the regions distal from splice sites, not surrounding m6A consensus sites. Data are presented as means ± SEM, n = 3 biological replicates per cell line. *p < 0.05 (two-tailed Student’s t tests), unless otherwise stated.

Fig 4: FUBP1 Promotes Alternative Splicing of Cancer Driver Genes(A) rMATS 4.0.1 used for detection of alternative splicing events in FUBP1-null cells. Pie chart shows distribution of A5′-splice site (A5SS), A3′-splice site (A3SS), skipped exon (SE), retained intron (RI), and mutually exclusive exon (MXE) splice events.(B) Enrichment of functions of alternatively spliced genes detected by rMATS, performed by DAVID functional annotation analysis.(C) Summary of cancer genes that are alternatively spliced in FUBP1-null cells, indicating gene name, biotype, and summary of function.(D–F) Western blots of NTC and FUBP1-null cell lysates for CASP8 (D), BRCA1 (E), and MAGI3 (F) and corresponding sashimi plot of alternative splicing. Blot for BRCA1 was stripped and re-probed for MAGI3. In sashimi plots, y axis represents a modified reads per kilobase of transcript (RPKM), per a million mapped reads. Peaks report number of junction reads. Below each, cartoon representations of alternative isoforms: exons and introns are not drawn to scale and represented as black rectangles and lines, respectively.

Fig 5: FUBP1 Loss Drives Several Characteristic Features of Transformation and, with PTEN Loss, Promotes Tumor Growth In vivo(A) Western blot of lysates from MCF10F cells transduced with NTC, PTEN, FUBP1, or PTEN+FUBP1 CRISPR-Cas9 sgRNA to show knockout of corresponding genes.(B) Proliferation of the indicated cell lines over 7 days, measured by MTS assay, analyzed with an ANOVA with a multiple-comparisons test.(C) Soft agar growth assays for the indicated cell lines. Images show representative soft agar fields for the indicated cell lines after 2 weeks. Scale bar represents 50 μm. Analyzed with one-way ANOVA with a multiple comparisons test against sgNTC.(D) Quantification of soft agar colonies in the indicated cell lines after 2 weeks.(E) Representative immunofluorescent (IF) images of the indicated cell lines after 10 days in hydrogels. Green, CK14+; red, CK8/18+. Nuclei stained with Hoechst (blue). Scale bars represent 100 μm. Pie charts represent quantification of 3D tissue morphology, cellular polarity, and expression. Blue sections represent T1 structures, red represent T2, green represent T3, and white represent T4.(F) Bioluminescence imaging was used to detect tumor growth in NOD-SCID mice injected with the indicated cell lines (n = 5 mice per cell line, 5 × 106 cells injected per gland).(G) Quantification of bioluminescence emitted from each injected gland at 2 weeks after injection.(H) Gross and microscopic detection of tumor growth in NOD-SCID gland injected with PTEN/FUBP1-null MCF10F cells. Images depict highly vascularized tumors (i) with angiogenesis and inflammation (ii and iii), as well as an abnormally mitotic cell (iv). Scale bars represent 50 μm. Data are presented as means ± SEM, n = 3 biological replicates per cell line. *p < 0.05, ***p < 0.0005 (two-tailed Student’s t tests unless otherwise indicated).