Fig. 6: IWS1 phosphorylation promotes cell proliferation by controlling a Sororin/ERK phosphorylation feedback loop, through U2AF2 RNA splicing. | Nature Communications

Fig. 6: IWS1 phosphorylation promotes cell proliferation by controlling a Sororin/ERK phosphorylation feedback loop, through U2AF2 RNA splicing.

From: RETRACTED ARTICLE: AKT3-mediated IWS1 phosphorylation promotes the proliferation of EGFR-mutant lung adenocarcinomas through cell cycle-regulated U2AF2 RNA splicing

Fig. 6

a, b Growth curves of shControl and shIWS1 NCI-H522 and NCI-H1299 cells, growing in fully supplemented media, before and after rescue with Sororin (wt or mutants), or U2AF65α/U2AF65β. Experiment was done in triplicate and proliferation was expressed as mean percent confluence ±SD. P values (one-sided unpaired t-test) were calculated for the endpoint measurements. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. b Growth curves of shControl and shIWS1 A549 and NCI-H1975 cells. c Growth curves of the same shIWS1 cells rescued with U2AF65α or U2AF65β. Proliferation in panels c and d was measured as in panels a and b. d (Upper panel) Cell cycle profiles of the indicated propidium iodide (PI)-stained cell lines. One representative, out of three biological replicates. Mean percentages of cells in different cell cycle phases in red. (Lower panel) shIWS1-induced percent change of cells in G2/M±SD. Statistical analysis was performed using a one-sided unpaired t-test, *p < 0.05. Gating strategy in supplementary figure 8a. e NCI-H1299 cells were sorted into G0/G1, S and G2/M fractions. Lysates of sorted cells were probed with the indicated antibodies. RT-PCR on RNA from the same cells, using primers for U2AF2 exons 1 and 3. Gating strategy in supplementary figure 8a. f qRT-PCR-determined expression of IWS1, CDCA5 and U2AF2, relative to GAPDH±SD. SD was calculated based on three biological replicates. g ChIC-determined mean fold enrichment of IWS1, SETD2 and H3K36me3 in cell cycle fractionated cells in the indicated regions of the U2AF2 RNA±SD. h qRT-PCR-based calculation of the mean U2AF2 E2/E3 ratio in S and G2/M, relative to G0/G1 cells ±SD. SD was calculated based on three biological replicates *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (one-sided unpaired t-test). i IWS1 expression is induced during the S and G2/M phases of the cell cycle. Following phosphorylation by AKT3, IWS1 orchestrates the cell cycle-dependent assembly of epigenetic complexes on the U2AF2 gene. This promotes the inclusion of exon 2 in the U2AF2 mRNA. U2AF65α, encoded by the exon 2-containing U2AF2 mRNA, interacts with Prp19, promoting CDCA5 splicing and the expression of Sororin. The latter is phosphorylated by ERK, and promotes ERK phosphorylation, in a positive feedback loop, which stimulates proliferation of lung adenocarcinomas.

Back to article page