Fig. 1: IWS1 expression and/or phosphorylation regulate alternative mRNA splicing. | Nature Communications

Fig. 1: IWS1 expression and/or phosphorylation regulate alternative mRNA splicing.

From: RETRACTED ARTICLE: AKT3-mediated IWS1 phosphorylation promotes the proliferation of EGFR-mutant lung adenocarcinomas through cell cycle-regulated U2AF2 RNA splicing

Fig. 1

a Western blots of NCI-H522 and NCI-H1299 cell lysates, transduced and probed with the indicated constructs and antibodies. b Overlaps between differentially-expressed and differentially spliced genes in the indicated groups (q < 0.05). c Bar graphs of alternative splicing events with exon inclusion. The comparisons were limited to alternative splicing events with a percentage of the alternatively spliced exon spliced in (psi/ψ) >0.6 and a p value < 0.05. d GO analysis of statistically significant alternative splicing events in the indicated groups (p < 0.05). Red boxes highlight gene sets involved in the regulation of RNA processing. e Volcano plots of all the exon inclusion and exclusion alternative splicing events, detected by DEXseq in the indicated groups. The statistically significant events (p < 0.05) with a percentage spliced in (psi/ψ) level of >0.6 or <0.4 are shown in red. Statistically significant events in the GO functions RNA splicing or RNA metabolic processes are shown in green. Alternatively spliced IWS1 targets validated in this report are shown in blue. f (Upper panel) RT-PCR of U2AF2 in the indicated NCI-H522 and NCI-H1299 cells, using primers mapping in exons 1 and 3, 3 and 5, and 8 and 10 (control). GAPDH was used as the loading control. The U2AF2 E2/E3 ratio was calculated from the GAPDH-normalized levels of the RT-PCR products. The bars show the mean ratio ± SD in the indicated NCI-H522 and NCI-H1299 cells relative to shControl. g Sequencing chromatograms of the two alternatively spliced U2AF2 RNA transcripts. h (Upper) UCSC browser snapshot showing exons 1, 2, and 3 of the human U2AF2 gene. The map position of the PCR primer sets used in the ChIP experiments in this figure is indicated by blue marks. (Lower) ChIP assays of IWS1 on the U2AF2 and GAPDH genes in shControl, shIWS1 shIWS1/WT-R and shIWS1/MT-R NCI-H522 and NCI-H1299 cells. Bars show the mean fold enrichment (anti-IWS1 IP, vs IgG control IP) in IWS1 binding, in shIWS1 relative to shControl cells or in shIWS1/MT-R relative to shIWS1/WT-R cells ±SD. Data were normalized relative to the input (2%). All assays were done in triplicate, on three biological replicates. n.s: non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (one-sided unpaired t-test).

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