Figure 2.

CAFs enhance the mesenchymal phenotype in PC9 cells.

Notes: (A) Representative images of NSCLC PC9 cells stably expressing GFP. Cells were stained with antibody targeting SMO (red), and nuclei were counterstained blue with DAPI. Scale bars: 20 µm. (B) Quantitative real-time RT-PCR analysis of human NSCLC PC9 cells grown in direct coculture with CAFs for 3 days at an erlotinib concentration of 1 µM. Data shown are representative of two independent experiments, and values represent the mean ± SD of triplicate samples. The expression of each mRNA was normalized to that of GAPDH mRNA in the same sample and is presented as the fold-change over that of vehicle-treated monoculture control cells. Difference in expression levels was evaluated for significance using one-sided Student’s t-tests with unequal variance (P<0.05). (C) Western blotting analysis showing the expression of genes regulating EMT and cell cycle. Cells were processed as (B) and probed with each antibody. GAPDH was used to show equal loading of protein.

Abbreviations: Mono-C, monoculture; Co-C, coculture; TGFβ, transforming growth factor-beta; EMT, epithelial to mesenchymal transition; CAF, cancer-associated fibroblast; NSCLC, non-small cell lung cancer; SD, standard deviation; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mRNA, messenger RNA; GFP, green fluorescence protein; SMO, smoothened; DAPI, 4′,6-diamidino-2-phenylindole dihydrochloride; RT-PCR, reverse transcription-polymerase chain reaction.