Fig 1: SCNN1B suppressed CRC growth in vivo.A DLD1-vector and DLD1-SCNN1B cells were subcutaneously implanted into nude mice. Representative images and tumor growth curve indicated that SCNN1B impaired the growth of tumor xenografts in vivo. B Images of tumors at the end point. Tumor weight was significantly reduced in SCNN1B overexpression group. C SCNN1B overexpression suppressed growth of SW1116 xenografts in nude mice. Representative images of tumors at the end point (left). The tumor volume (middle) and weight (right) were significantly reduced in SCNN1B overexpression group. D SCNN1B suppressed cell proliferation in DLD1 xenografts as determined by Ki-67 staining. E SCNN1B induced apoptosis in DLD1 xenografts as determined by TUNEL assay. F RT-PCR and Western blot demonstrated the successful overexpression of SCNN1B in tumor xenografts. G SCNN1B inactivated c-Raf in vivo, as indicated by increased S259 but reduced S338 phosphorylation. Values represent means ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.0001.
Fig 2: 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.
Fig 3: Re-expression of c-Raf reversed tumor suppressive effect of SCNN1B.A The re-expression of wildtype c-Raf and mutant c-Raf (S29A, S259A) in DLD1-vector and DLD1-SCNN1B cells. B SCNN1B overexpression suppressed the protein expression of S259A mutant c-Raf. C Addition of MG132, but not chloroquine, rescued c-Raf S259A protein expression in SCNN1B-expressing DLD1 cells (left). SCNN1B increased the ubiquitination of c-Raf S259A, but had no effect of wildtype c-Raf (right). D Ectopic expression of wildtype or mutant c-Raf had no effects on growth of DLD1-vector cells. For SCNN1B-overexpressing cells, wildtype or S29A c-Raf rescued cell proliferation. E In DLD1 and F SW1116 cells, overexpression of wildtype or S29A c-Raf reversed the inhibitory effect of SCNN1B on colony formation, but c-Raf S259A had no effect. G Re-expression of wildtype c-Raf, but not c-Raf S259A, rescued c-Raf kinase activity in SCNN1B-overexpressing DLD1 cells. H SCNN1B overexpression increased 48h-IC50 values of 5-Fluorouracil. I SCNN1B overexpression promoted apoptosis induction by 5-Fluorouracil. Values represent means ± S.E.M.*P < 0.05, **P < 0.01, ***P < 0.0001.
Fig 4: SCNN1B is silenced in CRC and is associated with patient survival.A Volcano plot of RNA-sequencing dataset from the Cancer Genome Atlas (TCGA) CRC cohort, showing that SCNN1B is an outlier gene down-regulated in CRC. B SCNN1B mRNA is up-regulated in TCGA CRC dataset in both paired samples (P < 0.0001) and the overall cohort (P < 0.0001). C RT-PCR and western blot validated that SCNN1B mRNA and protein are silenced in CRC tissues compared to adjacent normal colon tissues (left). qPCR validation in paired CRC and adjacent normal samples (right). D Immunohistochemistry staining showed that SCNN1B is down-regulated in CRC compared to adjacent normal samples. E Tissue microarray (TMA) CRC cohort for SCNN1B protein expression. Representative Kaplan-Meier survival plots of SCNN1B expression in CRC revealed that high SCNN1B expression is correlated with improved survival in the overall cohort (left), early-stage (middle), and late-stage CRC (right). F Cox-regression including univariate and multivariate analysis demonstrated that SCNN1B is an independent predictor of favorable survival in CRC patients.
Fig 5: SCNN1B functions as a tumor suppressor in CRC cells via a cytokinetic effect.A Overexpression of SCNN1B in DLD1 and SW1116 cells. B SCNN1B overexpression suppressed colony formation and C cell growth in DLD1 and SW1116 cells. D SCNN1B ectopic expression induced both early and late apoptosis in DLD1 cells, and promoted early apoptosis in SW1116 cells. E Western blot of apoptosis and cell cycle markers. SCNN1B increased cleaved caspase-8, caspase-9, caspase-7, and PARP indicative of apoptosis. SCNN1B suppressed Cyclin D1, whilst inducing p53, p21 and p27. SCNN1B inhibited MMP9, a marker for cell migration. F SCNN1B promoted G1-S cell cycle arrest in DLD1 and SW1116 cells, as evidenced by increased cells in G1 phase and decreased proportion of cells in S phase. G SCNN1B overexpression suppressed cell migration, as determined by wound healing assay. Values represent means ± S.E.M. *P < 0.05, **P < 0.01, ***P < 0.0001.
Supplier Page from MilliporeSigma for Anti-SCNN1B antibody produced in rabbit