Fig. 4
cNFIB inactivates ERK/MAPK signaling pathway. (A) Western blot analysis showed the expression of critical members of the MAPK signaling pathway in the indicated cells. (B) Left, representative images of immunohistochemical (IHC) staining of p-ERK in ICC tissues with low or high cNFIB expression. Right, the correlation between p-ERK and cNFIB levels of tumor tissues from 80 ICC patients; Pearson’s correlation test was used. (C) qRT-PCR analysis for the expression of downstream targets of ERK signaling in the indicated HuCCT1 (cNFIB knockdown) and RBE (cNFIB overexpression) cells. (D) Western blot analysis showing the expression of p-ERK and t-ERK in HuCCT1 cells transfected with ERK siRNA or RBE cells transfected with vectors expressing wild type (wt) or constitutively activated mutant ERK. (E) Western blot analysis showing the expression of p-ERK and t-ERK in HuCCT1 cells co-transfected with indicated siRNAs (left) and RBE cells co-transfected with indicated vectors (right). (F) qRT-PCR analysis for the expression of downstream targets of ERK signaling in the indicated cells. (G) CCK8 assays revealed cell proliferation capacity of the indicated cells co-transfected with indicated siRNAs or vectors. (H) Transwell assays showed the migration and invasion capacity of the indicated cells. Data were shown as mean ± SD, unpaired Student’s t test, *P < 0.05; **P < 0.01; ***P < 0.001. ERK2 (wt) was a plasmid expressing wild-type ERK2; ERK2L73P/S151D was a plasmid expressing constitutively activated mutant ERK2. Abbreviations: p-ERK, phosphorylation of ERK; t-ERK, total ERK