Figure 1.
Expression of EZH2 in NSCLC cells and the down-regulation of EZH2 by siRNA.
(A) EZH2 expression in NSCLC lung cancer cells and human bronchial epithelia cells (HBEs) were determined by Western blot using whole cell lysate. The blot was first probed with anti-EZH2 antibody and re-probed with anti-GAPDH antibody to indicate the loading quantity. (B) Immunocytochemistry staining of cultured NSCLC cells. A549 and Calu-1 cells were fixed by 1% formaldehyde then stained with anti-EZH2 antibody, followed by detection with anti-mouse secondary antibody and DAB as chromogenic agent. Secondary-antibody only is used as negative control. (C) Example of down-regulation of EZH2 expression by siRNA transfection. H1299 and A549 cells were transfected with EZH2 targeting siRNA si-4916 and si-4917 siRNAs or a scrambled si-RNA control. The whole cell lysate were harvested 72 hours post transfection and analyzed as described in A.
Figure 2.
The effect of EZH2 down-regulation on the growth of NSCLC cells.
(A, B) Proliferation plot of cells with down-regulated EZH2. A549 or 1299 were transfected with EZH2-targeting siRNA or scrambled siRNA control for 6 hours, then seeded into 96-well plate in triplicate per treatment. The proliferation of the cells was assessed by WST-1 assay at 24 hours interval. (C, D) Distribution of cell population in EZH2 knockdown NSCLC cells. A549 or 1299 cell were transfected with siRNA or scrambled control. The cells were harvested 72 hours later, fixed and stained with propidium iodide. Cellular DNA content were determined by flow cytometry and the distribution of cells is analyzed by FlowJo software.
Figure 3.
The effect of EZH2 down-regulation on anchorage-independent growth and invasion of NSCLC cells.
A549 or 1299 were transfected with EZH2-targeting siRNA or scrambled siRNA control for 6 hours. (A) For anchorage-independent colony formation assay, the transfected cells were seeded in 0.35% agarose in a 6-well plate at 2×104/well. The colonies formed were counted after 3-weeks incubated with regular change of medium. (B) For invasion and migration assay, 1×105 transfected cells in 1% serum were seeded on top of a BD BioCoat Matrigel invasion chamber with 10% FBS in the lower chamber. After 24 hours, the cells were fixed with 4% formaldehyde with 0.5% crystal violet. Cells invaded to the lower chamber were counted under low magnification microscope. The experiments were run in duplicate and repeated three time. The average number and standard derivation of each treatment were graphed here.
Table 1.
Demographic Characteristics of the Patient Population by EZH2 Expression.
Table 2.
Univariate and multivariate Cox proportional Hazard Model analysis for clinical and pathological factors that may affect survival after resection of NSCLC.
Figure 4.
Immnoblot assessment of EZH2 down-regulation on the expression of cell cycle regulators and apoptotic markers.
H1299 (A) or A549 (B) cells were transfected with EZH2-targeting siRNA or scrambled siRNA control as indicated. 72 hours post transfection, the cells were harvested and total protein was extracted. Twenty micrograms of protein per well were separated on SDS-PAGE and transferred to nitrocellulose membrane. The blot was probed with the indicated antibody and visualized by HRP-conjugated secondary antibody and ECL. Images on film were scanned using Canon film scanner. The expression level were quantified by digitized pixel density count using Adobe Photoshop and expressed as fold changes beneath the images.
Figure 5.
Probability of survival by EZH2 expression level.
Kaplan- Meier estimation of overall survival, disease-specific survival and disease-free survival by (A) EZH2 expression in tumor tissues relative to adjacent normal lung tissues (EZH2-fold change); and (B) EZH2 expression levels in tumor tissue by itself (dCt_Tumor). The median of each measure was used to dichotomize the patient population.