Fig. 2.

Promotion of epithelial-to-mesenchymal transition (EMT) by the knockdown of G protein subunit alpha Q (GNAQ). a) Expression of EMT markers. Epithelial and mesenchymal markers were determined by western blotting. β-tubulin is used as an internal control. b) Representative images demonstrating the expression of N-cadherin in control (A549_shNC) or GNAQ-knockdown (A549_shGNAQ) cells. c) Protein kinase B (Akt) and nuclear factor-κB (NF-κB) expression was determined by western blotting. β-tubulin served as a loading control. d) Representative microscopy images of migrating quantified by haematoxylin and eosin (H&E) staining. Transwell assays were conducted to detect cell migration. Significant differences are marked by asterisks (p = 0.014, two-tailed independent-samples t-test). e) Representative images of lung metastases monitored using bioluminescent signal imaging (left panel, n = four per group). Imaging times vary due to saturation at later acquisition time. Representative H&E-stained images of mouse lung (right panel). Arrows indicate metastatic nodules in the lungs. Scale bars, 500  μm. A549_shGNAQ_luc cells metastasized to other bone sites (bottom, left panel) (○ indicates bone metastasis site). Representative images of human mitochondria level (indicated by the arrows) by immunohistochemistry (IHC) of the bone (bottom, right panel); scale bars, 100 μm. Mice were euthanized 60 days after tibia injection with A549_shNC_luc and A549_shGNAQ_luc cells. DAPI, 4′,6-diamidino-2-phenylindole. shNC, negative control short hairpin RNA.