Agar plates were prepared by mixing complete growth medium with 2% noble agar to produce a solution of 0.7% agar. Agar solution (1.5 ml) was added to a well of a sterile six-well tissue culture plate and incubated at room temperature for 30 min to set. A549 (4 × 104 cells per well) and wild-type H1975 (1 × 105 cells per well) were mixed with preheated 2% noble agar to make a 0.3% agar solution, and 1.5 ml of the solution was added to each well on top of the bottom agar layer. One milliliter of fresh growth medium was then added over the top of the agar solution and replaced every 2 to 3 days. A549 cells were grown for 2 weeks, and wild-type H1975 cells were grown for 3 weeks, after which colonies were fixed and stained for 30 min in a 0.5% crystal violet/20% methanol/PBS. Colonies were imaged on an Olympus IX71 inverted fluorescence microscope (4× objective) and analyzed using ImageJ. Colony number and size were calculated in ImageJ.

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