Fig 2: ILF2 is highly expressed in NSCLC cell lines and is associated with poor patient outcome. (A) Representative gel presenting mRNA expression of ILF2 in HUVEC-C, HBEC-5i, BEAS-2B, A549, H460, H1155 and H1299 cell lines. (B) Protein levels of ILF2 were measured by western blotting in HUVEC-C, HBEC-5i, BEAS-2B, A549, H460, H1155 and H1299 cell lines. (C) Kaplan-Meier survival analysis of the association between survival time and ILF2 signature in lung cancer using an online tool (http://kmplot.com/analysis/). ILF2, interleukin enhancer binding factor 2.

Fig 3: ILF2 reduces cell-matrix adhesion and promotes anchorage independence. (A) Western blotting confirmed that ILF2 was successfully overexpressed in HUVEC-C and HBEC-5i cell lines. (B) Overexpression of ILF2 reduced the adhesion ability of HUVEC-C cells. Scale bars, 200 µm. (C) Resistance to anoikis was increased in suspended HUVEC-C cells following ILF2 overexpression. Results from same assay in HBEC-5i were similar (data not shown). *P<0.05 vs. control. (D) Western blotting confirmed successful knockdown of ILF2 in A549 and H460 cell lines. (E) ILF2 knockdown increased adhesion ability of A549 cells. Scale bars, 200 µm. (F) Ratio of suspended A549 cells undergoing anoikis was increased following ILF2 knockdown. Results from same assays in H460 were similar (data not shown). *P<0.05 vs. con shRNA. Attached, cells attached to the extracellular matrix; floating, cells suspended without the extracellular matrix; con, control; ILF2, interleukin enhancer binding factor 2; NS, not significant; sh, short hairpin.

Fig 4: DDX5 is an essential regulator of splicing in undifferentiated spermatogonia. a Gene ontology (GO) term enrichment analysis of data obtained from DDX5 immunoprecipitation followed by mass spectrometry using wildtype murine cultured undifferentiated spermatogonia (n = 3 independent experiments). b DDX5 immunoprecipitation followed by western blot in wildtype cultured spermatogonia confirming the interaction of DDX5 with proteins involved in pre-mRNA splicing (SRSF3), maintenance of mRNA stability (ELAVL1, PABP1, ILF3, ILF2, IGF2BP3), mRNA export (PABP1, SRSF3, ILF2, ILF3, IGF2BP3) and translation (IGF2BP3). Representative of n = 2 independent experiments. c Summary of differential splicing analysis performed between control and Ddx5-ablated cultured spermatogonia. Numbers of predicted alternative splicing events in each category upon Ddx5 deletion are indicated. d Visualisation of differential splicing analysis of RNA-sequencing data comparing control and Ddx5-ablated spermatogonia. Tracks are shown for selected candidate genes (left). Red arrows indicate differentially spliced exon. Schematics of alternative splicing events are shown (blue and yellow rectangles) (middle). Change in “percent spliced in” between conditions is shown as a value below splicing schematics (?PSI) and in bar charts (right). #: changes meet analysis cut-offs (?PSI >0.20, Bayes Factor =10). PSI ± 95% confidence interval shown. e RNA-immunoprecipitation using DDX5 antibody in cultured wildtype spermatogonia followed by PCR and gel electrophoresis for differentially spliced candidates depicted in d. Representative of n = 3 independent samples per condition. f PCR validation of differentially spliced candidates identified in d. CTL vehicle-treated control spermatogonia, KO Ddx5-ablated cultured spermatogonia. Differentially spliced exons are depicted by “?exon number”. Representative of n = 3 independent samples. Position of the PCR primers used are depicted in d in orange beneath schematic showing gene structure. g Bar chart of RNA-sequencing analysis for selected candidates. Fold change in Ddx5-ablated spermatogonia relative to vehicle-treated control. # denotes FDR <0.05. Mean ± SEM shown; n = 4 independent biological replicates per condition. h RAD50 protein levels by western blot in vehicle-treated control (Control) versus Ddx5-ablated cultured spermatogonia (Ddx5TAM-KO). *P < 0.05; two-tailed unpaired t-test; mean ± SEM shown; n = 4 independent biological replicates per condition. Representative western blot shown on right. i RAD50 immunofluorescence of vehicle-treated control (top) and Ddx5-ablated (bottom) cultured spermatogonia at D2 post-treatment. Representative of n = 4 independent biological replicates per condition. Scale bars = 100 µm

Fig 5: ILF2 can directly bind to the PTEN gene to regulate its transcription. (A) The seven regions designed for ChIP are highlighted. (B) ChIP analysis demonstrated that ILF2 antibody was enriched in the region R3 of PTEN in HUVEC-C and HBEC-5i cell lines following ILF2 overexpression, according to RT-qPCR results. (C) Representative gel for R3 of PTEN following ChIP analysis of HUVEC-C and HBEC-5i cell lines that overexpressed ILF2, according to RT-qPCR results. (D) Luciferase reporter studies indicated that the region of PTEN regulated by ILF2 may be located within the −2956 to −2642 bp fragment. Asterisks indicate site with ILF2 consensus sequences. ChIP, chromatin immunoprecipitation; Ctrl, control; IgG, immunoglobulin G; ILF2, interleukin enhancer binding factor 2; PTEN, phosphatase and tensin homolog deleted on chromosome ten; R3, region 3; TSS, transcriptional start site; Ctrl, control.