Fig 2: Effects of overexpression of FGFR1α and FGFR1β in MCF-10A cells on FGFR signaling and cell proliferation(A) Viral FGFR1 expression. Virus particles were packaged for FGFR1α and FGFR1β, and were infected into MCF-10A cells. Expression of FGFR1 was confirmed by immunoblotting with anti-FGFR1 antibody. (B, C) Immunoblotting of FGFR signaling activity. MCF-10Acells were treated with FGF2 at 20ng/ml or vehicle control for 24 hours. The MAPK pathway was detected in the cell lysates with antibodies against phospho-MEK1/2, phospho-ERK1/2 (B), while the PI3K pathway was detected with antibodies against phospho-pAKT, phospho-S6 and phospho-4E-BP1 (C). (D) Cell growth rate. MCF-10A cells expressing FGFR1α, FGFR1β, and vector were cultured in 96-well plates in a normal condition for the indicated days. Cell viability was measured by SRB staining. Cell proliferation rate was calculated by normalizing OD490nm values to day 1 OD value. #: p<0.05 vs empty vector; *: p<0.01 vs empty vector. (E-G) Cell colony formation. MCF-10A cells seeded in 6-well plates were cultured for 3 weeks followed by crystal violet staining (E). Total colony area (F) and average colony size (G) were quantitated using ImageJ software. Average values were calculated from triplicate wells for each group. *: p<0.01 vs empty vector.

Fig 3: EGFR/MEK/ERK pathway activation is involved in RNF128 enhanced HCC progression: (A) mRNA expression level correlation between RNF128 and EGFR/MEK/ERK pathway marker genes (EGFR, MAP2K1, MAPK1) in HCC cells. (B) Expression of EGFR/MEK/ERK pathway markers (p-MEK, MEK, p-ERK, ERK, EGFR, p-EGFR) in MHCC97-L-control/MHCC97-L-shRNF128 and HepG2-control/HepG2-RNF128 cells quantified by Western blotting. (C) RNF128 over-expressing cells were pretreated with gefitinib (1μM) for 24 hours, Western blot analysis of EGFR/MEK/ERK pathway markers (EGFR, p-EGFR, MEK, p-MEK, ERK, p-ERK) expression in the indicated cells. Gefitinib counteracted RNF128-enhanced p-EGFR, p-ERK, p-MEK expression in HepG2 cells. (D and E) RNF128-enhanced migration, invasion, and proliferation in HepG2 cells was eliminated by gefitinib. Scale bar: 200× = 100μm. *p<0.05, **p<0.01; ***p<0.001.

Fig 4: The MAPK pathway is involved in CCL28-mediated tumor metastasis. (A) Western blot analysis of MMP2, MMP9, VEGF and β-catenin expression in MDA-MB-231HM/vector (231HM/vector) and MDA-MB-231HM/CCL28 (231HM/CCL28) cells. (B) β-catenin protein expression was detected by immunohistochemical analysis in xenografts of 231HM/vector and 231HM/CCL28 tumor-bearing mice. (C) Association of CCL28 with the MAPK signaling pathway. Overexpression of CCL28 increased the level of phosphorylated ERK1/2, MEK1/2, and JNK1/2 as determined by western blot analysis. (D) Immunohistochemical examination of pERK protein expression in xenografts of 231HM/vector and 231HM/CCL28 tumor-bearing mice. MAPK, mitogen-activated protein kinase; MMP, matrix metalloproteinase; VEGF, vascular endothelial growth factor.

Fig 5: Paradigm describing how differences in MAPK signaling lead to inter-individual variability in phBEC ozone-mediated IL-8 induction. Previous studies have suggested that ozone exposure leads to the activation of the MAP Kinases ERK1/2 and p38 by various mechanisms including the activation of the EGFR receptor. The activation of both kinases is important for full ozone-mediated IL-8 induction, yet the magnitude of ozone-induced IL-8 expression is only correlated with the level of ERK1/2 activation. Based on these observations, we propose a ‘dimmer switch’ model describing how p38 and ERK1/2 act in concert to regulate IL-8 expression. A basal level of p38 activation is required to achieve full IL-8 expression (acting as an ‘on/off’ switch), while the level of ERK1/2 pathway activation modulates the magnitude of expression (the ‘dimmer’). These kinases both activate effector proteins and kinases such as RSK and MK2, in addition to other transcription factors and stress-associated proteins. These ultimately converge on the IL-8 promoter and regulate gene transcription.