Deficiency of Cdkn2a leads to aberrant activation of RhoA in vivo.

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Deficiency of Cdkn2a leads to aberrant activation of RhoA in vivo. Deficiency of Cdkn2a leads to aberrant activation of RhoA in vivo. A, hematoxylin and eosin (H&E) staining of lung tissue sections from the indicated genotype after 12 weeks of KrasG12D induction. Left, low magnification scale bars, 1 mm; right, high magnification scale bar, 40 μm. B, top, tumor number and grade/mouse in KrasG12D;Ink4a/Arf−/− and KrasG12D;Ink4a/Arf+/+ lungs after 12 weeks of KrasG12D induction. The average of 3 representative lung tissue sections/mouse were analyzed (n = 8/genotype). A, lung adenoma; AC, lung adenocarcinoma. Bottom, representative H&E image of a mixed KrasG12D;Ink4a/Arf−/− tumor (exhibiting both low- and high-grade features). Scale bar, 40 μm. C, immunoblot of microdissected lung adenomas of KrasG12D;Ink4a/Arf+/+ mice and adenocarcinomas of KrasG12D;Ink4a/Arf−/− mice obtained after 4, 8, or 12 weeks of KrasG12D induction. Each lane represents a lysate from a single mouse. D, p-Erk1/2 (Thr202/Tyr204) IHC staining of representative lung tumors of the indicated genotype after 12 weeks of exposure to doxycycline. An adjacent H&E–stained section is shown to illustrate areas of adenocarcinomas (AC) and adenomas (A). Inserts indicate area selected for higher magnification shown below. Top, scale bar: 100 μm. Bottom, scale bar, 40 μm. Red and black arrowheads indicate adenocarcinomas and adenomas respectively. E, representative immunoblots showing Ras-GTP levels along with p-Erk-Rac1-RhoA activation levels. From top to bottom: p-Erk1/2 followed by GST-PAK1, GST-RBD, and GST-RAF1 pulldown for Rac1-GTP, RhoA-GTP, and Ras-GTP respectively, from microdissected lung tumors of the indicated genotypes. Each lane represents a lysate from a single mouse. F, top, H&E staining of a lung tumor section to show areas of adenomas (A) and adenocarcinomas (AC) from KrasG12D;Ink4a/Arf−/− mice. Ovals show higher-magnification images of sections stained with H&E. Bottom, RhoA-GTP IHC staining of lung (12 weeks KrasG12D) tumors. Dotted lines demarcate A (adenomas) and AC (adenocarcinomas). Left, lower magnification; scale bars, 100 μm; right, higher magnifications; scale bars, 40 μm. Inserts and arrows indicate areas magnified further on the right panels. Georgia Konstantinidou et al. Cancer Discovery 2013;3:444-457 ©2013 by American Association for Cancer Research