Fig. 4: SCNN1B suppressed MAPK signaling via c-Raf-MEK-ERK cascade.

A Gene set enrichment analysis (GSEA) with Cytoscape enrichmentMap revealed SCNN1B expression closely correlates with oncogenic KRAS signature in TCGA CRC RNA-seq dataset. B Enrichment plots showing that SCNN1B expression enriched for genes down-regulated by oncogenic KRAS. C MAPK antibody array analysis of DLD1 cells transfected with empty vector or SCNN1B demonstrated that SCNN1B down-regulated MAPK signaling, especially p-ERK1/2 and p-AKT. D MAPK signaling PCR array analysis of DLD1-vector and DLD1-SCNN1B cells revealed a consistent down-regulation of MAPK downstream genes, including cyclins and kinases. E DLD1, SW1116, and AGS cells expressing SCNN1B exhibited down-regulated SRE luciferase activity, an indicator of MAPK signaling. F Western blot validated that the overexpression of SCNN1B suppressed the phosphorylation (active) of AKT1, AKT2, MEK, and ERK, in agreement with the antibody and PCR data results. G Active KRAS pull-down assay revealed that SCNN1B had no effect on active KRAS activity. H c-Raf kinase activity was suppressed by SCNN1B overexpression in DLD1 and SW1116 cells. I Western blot of c-Raf, A-Raf, B-Raf, and their respective phosphorylated forms. SCNN1B induced the phosphorylation of c-Raf at S259 and S289, both of which are inactivating phosphorylation. It also reduced c-Raf S338 (active) phosphorylation. Besides, SCNN1B had no consistent effect on B-Raf or A-Raf expression and phosphorylation. Values represent means ± S.E.M.*P < 0.05, **P < 0.01, ***P < 0.0001.