PTC-209 is a potent and selective BMI-1 inhibitor.

BMI1:0.5 μM

PTC-209能降低体内功能性的结直肠癌CICs的频率.PTC-209（60 mg/kg/day,s.c.）有效地抑制肿瘤组织中BMI-1的产生,并停止在具有原代人结肠癌异种移植物,人结肠癌细胞系LIM1215或HCT116异种移植物的小鼠中预先建立的肿瘤的生长.

PTC-209通过不可逆的生长抑制破坏结肠直肠癌起始细胞（CIC）。PTC-209抑制人结肠直肠HCT116和人纤维肉瘤HT1080肿瘤细胞中UTR介导的报道基因表达和内源性BMI-1表达。PTC-209以BMI-1依赖性减少直肠肿瘤细胞的生长。

Untranslated region-mediated luciferase reporter expression: HEK293 cells are transfected with a GEMS reporter vector that contains the luciferase open-reading frame flanked by and under post-transcriptional control of the BMI-1 5′ and 3′ UTRs. The resulting stable cells (F8) are treated with PTC-209 or vehicle control overnight, and then luciferase reporter activity is determined using Bright-Glo assays. The assays are run in triplicate for each point, and the percentage of inhibition was calculated against vehicle control.

To determine whether pretreatment with the inhibitor affects tumor cell growth, cells are plated with the inhibitor for 4 d in vitro and plated in limiting doses in vitro without adding further inhibitor. Trypan blue exclusion is used to count viable cells. The in vitro sphere-initiating cell frequency is calculated after inhibitor treatment by evaluating the number of wells containing spheres. For the experiments where LDAs are set up following recovery of PTC-209 treated cells, 6-well plates were seeded with 1E6 cells per well and incubated overnight. Cells are subsequently treated for 4 d in triplicate with either DMSO vehicle or PTC-209 (0.01, 0.1, 1 and 10 μM). Drug treatments are washed off and 4 mL fresh suspension medium added to all wells. To assess cell viability following the 4 d treatment window, cells are trypsinized and counted at 0, 24, 72 and 120 h after removal of the drug. Long-lasting effects of the drug treatment on sphere-forming ability are assessed by plating LDAs (50,000, 10,000, 1,000,100, 10 and 1 cell per well) using the cells obtained 120 h after the 4-d drug treatment.(Only for Reference)