Cell density affects experimental outcomes.

(A) HEK 293FT cells were plated in 6 well-plates according to the following scheme: 5 sets of control cells at a range of densities (100-500K cells per well) and 4 sets of cells at the highest density (500K cells per well) for treatment. All control cells were treated with the vehicle (control), and the remaining 4 sets were treated with 25 μM Ly294002 (Ly), 2.5 μg/μl Brefeldin A (BFA), 100 ng/ml nocodazole (noco) or 800 nM YM-201636 (YM), for the last 20h before lysis. Ctl N, control cells plated at the same number (500K) as the treated cells; Ctl D, control cells with matching cell density at the experimental endpoint. In classical experimental design, Ctl N is used as a control. (B) Cells were imaged before the lysis and matched with one of the controls based on cell density. Scale bar, 100 μm. (C-F) Cell lysates were analyzed by Western blotting using indicated antibodies. Ctl N and Ctl D were simultaneously compared with treated cells. GAPDH was used as a loading control. (G-J) Western blot images were quantified and the values normalized to GAPDH; Bar graph data are mean ± SD. *p<0.05, **p<0.01, ***p<0.001.