Fig. 6

PKG activation by PA5 interferes with the EGF/EGFR pathway. SKMEL-28 and M121224 cells were serum starved for 24 hours, treated with PA5 for 4 hours, and then stimulated with EGF for 30 minutes. (a) Immunoblotting of p-EGFR, EGFR, p-Akt, p-ERK1/2 after exposure to EGF or to EGF together with PA5. (b) RQ of protein levels of p-EGFR and EGFR were calculated by ImageJ software using β-actin as a normalizer. (c) The p-EGFR-to-EGFR ratio was calculated using ImageJ data. (d) Protein levels (RQ) of p-Akt and p-ERK1/2 were calculated by ImageJ software using β-actin as a normalizer. (e) Immunoblotting of N-cadherin, Cyclin B1, and BIM proteins in M121224 cells 24 hours after EGF and PA5 treatment. (f) Proteins levels (RQ) of N-cadherin, Cyclin B1, and BIM were measured by ImageJ software using β-actin as a normalizer. Data represent the means ± SD of three independent biological experiments. Student’s t-test was used for statistical analyses, and P-value was calculated referring to the EGF level. ∗∗∗P < 0.001; ∗∗0.001 < P < 0.01; ∗0.01 < P < 0.05. (g) Schematic representation of the proposed molecular mechanism. ERK, extracellular signal‒regulated kinase; MEK, MAPK/extracellular signal‒regulated kinase; NT, not treated; PA, protein kinase G activator; p-Akt, phosphorylated protein kinase B; p-EGFR, phosphorylated EGFR; p-ERK, phosphorylated extracellular signal‒regulated kinase; PKG, protein kinase G; PI3K, phosphoinositide 3-kinase; RQ, relative quantity.