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. 2022 May 25;13(5):496. doi: 10.1038/s41419-022-04941-3

Fig. 4. MEKi results in STAT3 phosphorylation, which occurs through SOCS3 downregulation.

Fig. 4

A Western blotting shows the expression of pSTAT3 and pERK in KYSE30 WT, ERK1/2-DKO-1, and ERK1/2-DKO-2 after EGF (50 ng/ml), OSM (10 ng/ml), or IL-6 (10 ng/ml) treatment. B KYSE150 WT and ERK1/2-DKO cells were treated with EGF (50 ng/ml), OSM (10 ng/ml), or IL-6 (10 ng/ml) for 1 h. Expressions of pSTAT3 and pERK were determined using western blotting. C RNA-seq heat map analyses of differentially expressed genes (DEGs) involved in the JAK-STAT3 signaling pathway after trametinib or DMSO treatment. A fold change cutoff of log2 < −0.58 or >0.58 and a p value cutoff of P < 0.05 were selected and overlapped for JAK-STAT3 signaling pathway genes. D qRT-PCR results show the transcription level of SOCS3 mRNA after trametinib treatment at various time points. Error bars represent the mean ± SD. **P < 0.01, ***P < 0.001. E KYSE30 and KYSE150 cells were incubated with trametinib (1 μM) for 0, 1, 2, 4 h, immunoblotted for SOCS3 expression. F, G Western blotting results show the expression of SOCS3 after EGF (50 ng/ml) treatment in WT, ERK-DKO KYSE30 (F) and KYSE150 (G) cells. H KYSE30 ERK1/2-DKO cells stably expressing EV, ERK2-HA, and ERK2-K54R-HA were used for western blotting to determine SOCS3 expression. I Western blotting results show the expression of pSTAT3 and Flag-SOCS3 in KYSE30 and KYSE150 cells transduced with EV or Flag-SOCS3. Trametinib (1 μM) treatment was applied at various time points. J Western blotting results show the expression of pSTAT3 and pERK1/2 in KYSE30 and KYSE150 cells infected with shNC or shSOCS3 lentiviral with two target sites.