Figure 2.

Establishing the miR-34a Inducible Expression System and an Assay for the Small Molecule Screen

(A) Schematic of the cell line system for miR-34a expression inducible by adding doxycycline. The miR-34a pre-miRNA was placed next to a TET responsive element, and CopGFP was placed under control of EF1a core promoter. Cells were selected for GFP expression and used in downstream assays.

(B) Expression of miR-34a in A549-miR-34a cells after adding increasing doses of doxycycline (DOX) for 48 h (***p < 0.005, Student's t test).

(C) miR-34a reporter luciferase activity upon addition of doxycycline at 1 μg/mL dose to A549-miR-34a cells, compared with non-transfected (NT) cells or without DOX, time point set to 48 h (***p < 0.005, Student's t test).

(D) Expression of miR-34a downstream genes CD44 and SRC after inducing miR-34a expression using DOX at 1 μg/mL treatment with time course monitored up to 72 h (*p < 0.05, **p < 0.01, Student's t test). Bars and errors bars represent the means and the corresponding SEMs for n ≥ 3.

(E) Establishing the cell-based assay for detection of total cell counts and caspase 3-activated cells. DOX and Erlotinib were added to a set of A549-miR-34a cells plated in parallel, and total cell numbers and activated caspase 3-positive cell counts were taken, followed with data analysis to measure the Z-factor.