Fig. 2: IWS1 phosphorylation-dependent mRNA splicing of U2AF2 is regulated by serum and IGF-1-induced signals, transduced by AKT3. | Nature Communications

Fig. 2: IWS1 phosphorylation-dependent mRNA splicing of U2AF2 is regulated by serum and IGF-1-induced signals, transduced by AKT3.

From: RETRACTED ARTICLE: AKT3-mediated IWS1 phosphorylation promotes the proliferation of EGFR-mutant lung adenocarcinomas through cell cycle-regulated U2AF2 RNA splicing

Fig. 2

a (Upper panel) Following serum starvation for 24 h, NCI-H522 and NC-H1299 cells were stimulated with IGF-1 or harvested. Four hours later, the stimulated cells were lysed and all the lysates were probed with the indicated antibodies. (Middle panel) RT-PCR with mRNA derived from the cells shown in the upper panel and oligonucleotide primers mapping to exons 1 and 3 of the U2AF2 gene (Lower panel) The U2AF2 gene E2/E3 ratio was calculated following quantification of the RT-PCR products in the middle panel. The bars show this ratio (mean± SD) in IGF1-stimulated and in unstimulated, serum-starved NCI-H522 and NCI-H1299 cells. b (Upper panel) Lysates of NCI-H522 and NCI-H1299 cells, treated with MK2206 (5μM) or DMSO for 4 h, were probed with the indicated antibodies. (Middle panel) RT-PCR reactions, using mRNA derived from the cells in the upper panel and oligonucleotide primers mapping in U2AF2 exons 1 and 3. (Lower panel) The U2AF2 mRNA E2/E3 ratio was calculated following quantification of the RT-PCR products in the middle panel. The bars show this ratio (mean± SD) in MK2206-treated (5μM) and in DMSO-treated NCI-H522 and NCI-H1299 cells. c (Upper panel) Lysates of NCI-H522 and NCI-H1299 cells, transduced with shControl, shAKT1, shAKT2, or shAKT3, were probed with the indicated antibodies. (Middle panel) RT-PCR reactions, using mRNA derived from the cells in the upper panel and oligonucleotide primers mapping in U2AF2 exons 1 and 3 show that knocking down AKT3, and to a lesser extent AKT1, inhibits the inclusion of exon 2 in mature U2AF2 mRNA transcripts in both cell lines. (Lower panel) The U2AF2 E2/E3 ratio was calculated following quantification of the RT-PCR products in the middle panel. The bars show this ratio (mean ± SD) in shControl, shAKT1, shAKT2 and shAKT3 NCI-H522 and NCI-H1299 cells. All experiments in this figure 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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