Knockdown of CDKN1B or CDKN1C reverses growth inhibition of TOX-sh cells. (A) CDKN1B knockdown by 2 shRNA (sh-1 and sh-2) in addition to TOX knockdown in Hut78 and HH cells. Transduced cells were selected by hygromycin (750 μg/mL for Hut78 and 200 μg/mL for HH) for 7 days before the protein lysates were probed with antibodies against p27 and actin proteins. (B) CDKN1C knockdown in addition to TOX knockdown in Hut78 and HH cells. Transduced cells were selected by hygromycin for 7 days before western blot analysis was performed to examine p57 protein levels. (C) Cosilencing of TOX with CDKN1B or CDKN1C led to increased proliferative rate in Hut78 cells. A total of 1.5 × 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. (D) Cosilencing of TOX with CDKN1B or CDKN1C led to increased proliferative rate in HH cells. Experiments were conducted in the same way as for Hut78 cells. *P < .05, **P < .01, and ***P < .001 by 2-tailed Student t test with Welch correction. Error bars indicate standard error of the mean. Data depicted are representative of at least 3 independent experiments.
