(A) Colony-forming assays showed a significant reduction in the number of colonies for the 5 CDX2-expressing cell lines SKM-1, MV4-11 (1 × 10⁴ plated cells), THP-1, MOLM-14, and NOMO-1 (1 × 10³ plated cells) after CDX2 mRNA knockdown by shRNA TRCN13684 as compared with cells transduced with the nonsilencing control construct. In contrast, transduction with shRNA TRCN13684 did not reduce colony formation of EOL-1, which had a very high CDX2 expression level (37,388; 82% mRNA knockdown; estimated residual expression level, 6,730); MONO-MAC-6, in which CDX2 expression was not efficiently silenced (4% mRNA knockdown); and HL-60 and K-562, which do not express CDX2 mRNA. Experiments were performed in duplicate. Values are represented as mean ± SEM. *P < 0.05; **P < 0.001. (B and C) Microscopic analysis of colonies derived from shRNA-transduced cells (right panels) showed a decrease in the number of colonies and the number of cells per colony, as compared with cells transduced with the nonsilencing control construct (left panels), for CDX2-expressing cell lines but not for CDX2-negative K-562 cells. Representative photomicrographs of methylcellulose cultures are shown. Original magnification, ×20 and ×100, respectively. (D) CDX2-expressing AML cell lines showed varying absolute mRNA levels after shRNA-mediated CDX2 knockdown.