(A) On a time-course MTT assay, A549G (squares) grew 4.3-fold and A549F (triangles) grew 2.6-fold faster than the empty nonsilenced A549 cells (circles, A549E; diamonds, A549SR). No differences were observed between A549E and A549SR. (B–D) Cells deficient in AHRR (A549F [C] and A549G [D]) showed a significant shift in the number of cells towards S and G₂/M phases as compared with the control A549E (B). Consistently, a reduction in the number of cells in G₀/G₁ phase was observed in A549F/G. (E and F) Artificial downregulation of AHRR enhanced A549 (E) and MCF10A (F) colony formation (60% and 300% increase in average colonies for A549F/G and A549E, respectively). MCF10A-F and MCF10A-G were able to clone in soft agar, while, as expected, MCF10A-E did not form colonies. All clonogenic assays were run in triplicate. (G and H) Comparison of the morphology of the colonies formed by A549E (G) and A549G (H). A549E grew in compact spheroids with defined contours. A549G colonies showed irregular morphology and cells detached and partially separated from the core of the colony. Original magnification, ×20. (I) In an in vivo experiment, A549 cells transfected with a siRNA for AHRR showed a significant increase in xenograft tumor volume 8 weeks after injection when compared with empty plasmid–transfected A549 cells (3-fold increase for A549F [squares] compared with A549E [triangles] and 7-fold increase for A549G [diamonds] compared with A549E). n = 10 animals/group. *P < 0.05; **P < 0.01; ***P < 0.001.