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. 2019 Dec 13;10:5712. doi: 10.1038/s41467-019-13392-y

Fig. 5. Functional significance of U2AF1 S34F in relation to SLC34A2-ROS1 fusions.

Fig. 5

a Gene set enrichment analysis (GSEA) plot of differential expression data. The most enriched gene set is the hallmark epithelial mesenchymal transition. b Relative protein expression of E-cadherin and fibronectin 1 (FN1) in HCC78 cells. Experiments were performed in triplicate. c Overexpression of U2AF1 S34F mutant increases invasion potential in HCC78. Wild-type and mutant U2AF1 were overexpressed in a doxycycline-inducible manner and plated for 48 h. d Schematic of SLC34A2-ROS1 splicing in HCC78. Exon 4 of SLC34A2 is fused to exon 32 or exon 34 of ROS1, creating long and short isoforms, respectively. e Relative expression of long:short SLC34A2-ROS1 isoform ratios in mutant and wild-type U2AF1 overexpressed cell lines as measured by RT-qPCR using isoform specific primers. The long and short isoforms in the mutant and wild-type overexpressed U2AF1 cell lines were normalized to the parental cell line, HCC78. f Relative protein expression of long to short SLC34A2-ROS1 isoforms in mutant and wild-type U2AF1 transduced cell lines as measured by Western blots, where HCC78 isogenic cells were exposed by 0.2 uµg/ml of doxicycline for 0, 1 day and 5 day to induce the U2AF1 expression. The long and short isoforms in the mutant and wild-type transduced U2AF1 cell lines were normalized to the GAPDH, with densitometry data from 5d summarized in bar plot (right). The band intensity was measured by Image J software. g The long ROS1 isoform increases invasion potential in the NIH-3T3 cell line after 48 h. The short and long SLC34A2-ROS1 isoforms were transduced in NIH-3T3 fibroblasts using lentivirus. h Sensitivity of wild-type versus S34F mutant U2AF1 transduced HCC78 cells to the ROS1 inhibitor crizotinib. Experiments were done with 6 technical replicates where cells were exposed to the drug for 24 h and cell survival rate was measured by manufacturer’s standard protocol (CellTiter-Glo® Luminescent Cell Viability Assay, Promega). i Proposed mechanism of U2AF1 S34F in LUADs with ROS1 fusions. U2AF1 S34F alters short and long SLC34A2-ROS1 isoform ratios. Error bars are used to illustrate the standard deviations around the means.