TOX suppression confers growth disadvantage to CTCL cells. (A) TOX knockdown by 2 shRNA (sh-1 and sh-2, both specific for TOX mRNA) compared to control (CTR) CTCL cells transduced by a nontargeting shRNA. Transduced cells were selected by puromycin (1 μg/mL) for 5 days before protein lysates were probed with antibodies against TOX and actin proteins. (B) TOX-sh cells had a much reduced viable cell number over a period of 4 days compared to CTR cells. A total of 2.5 × 105 cells were cultured in 2 mL of full RPMI media for 4 days, and viable cells were determined each day by the trypan blue exclusion method. (C) TOX-sh cells generated fewer and smaller colonies in 3-dimensional culture compared to CTR cells. Three hundred cells were plated in 1.5 mL of methycellulose, and colony numbers and morphology were recorded on day 12. Numbers of colonies of different sizes (big, medium, and small) are depicted. Photographs of representative colonies are also shown. Original magnification ×10. **P < .01 and ***P < .001 by 2-tailed Student t test with Welch correction. Error bars indicate standard error of the mean. Data shown here are representative of at least 3 independent experiments.
