(A-C) RKO cells, stably infected with lentiviruses expressing shRNA targeting luciferase (sh-Luc) or the SESN2 (sh-SESN2) gene, were subjected to colony-forming assay. In brief, cells were seeded sparsely (1000 cells per well for 6-well plates), treated with 5-fluorouracil (5-FU, 1 μM) and irinotecan (CPT-11, 10 μM) as indicated for 24 hr, and grown for 14 days in normal medium. Colonies were fixed in methanol and stained with 0.1% crystal violet (C581, Fisher Scientific). The plates were imaged under EPSON Perfection V30 scanner (A). Relative colony growth was determined by densitometry of stained images (B; n=3 from three different plates per group), and the growth suppression effects of 5-FU and CPT-11 were calculated from the densitometry results (C; n=3 from three different plates per group). (D,E) RKO cells, stably infected with sh-Luc or sh-SESN2 gene, were treated with indicated concentrations (μM) of 5-FU and CPT-11 for 24 hr and subjected to immunoblotting of indicated proteins. (F) Current model of how Sestrin2 suppresses colon cancer progression. During colitis, Sestrin2 promotes restoration of colon homeostasis after injury through limiting ER stress. However, during carcinogenesis, p53 is inactivated and Sestrin2 expression is downregulated, which subsequently causes hyperactivation of mTORC1 signaling and promotes colon cancer development and growth. The p53-Sestrin2 axis may also be important for the effect of chemotherapy in attenuating colon cancer growth. All data are shown as the mean ± s.e.m. *p<0.05, **p<0.01. P values are from Student’s t-test.