Fig. 2

PTC-209 reduced Bmi1 expression by transcriptional repression and protein degradation in HNSCC cells. a Endogenous Bmi1 protein was efficiently inhibited by PTC-209 in a time- and dosage-dependent manner in Cal27 and FaDu cells for indicated times. Representative images of WB are shown. b The mRNA levels of Bmi1 were significantly decreased following PTC-209 exposure (10 μM) for 48 h, and its well established downstream target p16 was derepressed. c The Bmi1 inhibition by PCT-209 treatment (10 μM, 48 h) was partially attenuated by pretreatment with the ubiquitin-proteinase inhibitor MG132 (5 μM, 6 h). Representative images are shown. d The ubiquitination of Bmi1 was measured in both cells treated with PTC-209 (10 μM, 24 h) followed by MG-132 (10 μM) for additional 6 h. Total cell lysates were immunoprecipitated with anti-Bmi1 antibody and then blotted for ubiquitin (Ub). GAPDH was used as a loading control. Representative images are shown. Data showed here are mean ± SD from three independent experiments, **p < 0.01, Student’s t test