Fig. 1: Effects of OST-01 on proliferation and apoptosis of AML cell lines and LSC-enriched AML blasts.

A Effects of OST-01 on proliferation and apoptosis of LSC-enriched AML blasts. CD34 + CD38- cells were isolated from primary MNCs (n = 5) or AML blasts (n = 4). Left, levels of cell proliferation. Right, levels of apoptosis. B Effects of OST-01 on colony forming of LSC-enriched AML blasts. CD34 + CD38- AML blasts or MNCs (2 × 10⁵ cells/ml, n = 3) were treated with 1 µL/mL of ethanol control or indicated dose of OST-01 for 24 h before plating on methylcellulose. After 14 days, colonies were images under light microscope and counted, N =  2, data are presented as mean ± SE, with triplicate determination. Number of colonies are presented as bar graph. Asterisk indicates statistically significant difference based on unpaired t test analysis. C Heatmap showing change of gene expression in primary CD34 + CD38- AML blasts treated with ethanol control or OST-01 (1 µL/mL) for 24 h (each group, n = 3). D Effects of OST-01 on expression of deubiquitinase USP36 and nucleoprotein. Primary CD34 + CD38- AML blasts were treated with ethanol control or dose dependent of OST01 (+0.5 µL/mL; ++1 µL/mL). Immunoblotting of indicated antibodies are shown. Quantification of protein expressions are shown on top. E Effects of OST-01 on cellular distribution of nucleolar proteins. Primary CD34 + CD38- AML blasts were treated with ethanol control or OST-01 (1 µL/mL) for 24 h, following by staining with anti-NPM1 and anti-nucleostemin antibodies. The images were taken under a confocal microscope. Scale bar, 10 µm. F Effects of OST-01 on nucleolus structure. Primary CD34 + CD38- AML blasts were treated with ethanol control or OST-01 (1 µL/mL) for 24 h. Transmission electron microscope (TEM) was performed to image nucleolus structure. Enlarged images are shown on the right. FC fibrillary center; DFC dense fibrillar component; GC granular component. G Effects of OST-01 on number of ribosome upon treatment of primary CD34 + CD38- AML blasts with ethanol control or OST-01 (1 µL/mL) for 24 h. Left, TEM images of ribosome. Right, quantification of ribosome levels. Asterisk indicates significantly different based on unpaired t test analysis. H Effects of OST-01 on ubiquitination of nucleoprotein. Primary CD34 + CD38- AML blasts were treated with ethanol control or OST-01 (1 µL/mL) for 24 h. The cell lysate was immunoprecipitated with anti-c-Myc, anti-NPM1 or anti-nucleostemin and immunoblotted with anti-Ubiquitin (Ub) antibodies. I Effects of USP36 knockdown on ubiquitination of c-Myc and NPM1 protein. Primary CD34 + CD38- AML blasts were transfected with scramble siRNA control or USP36 siRNA (40 nM) for 24 h. The cell lysate was immunoprecipitated with anti-c-Myc and anti-NPM1 and immunoblotted with anti-Ubiquitin (Ub) antibodies. J Effects of USP36 overexpression on OST-01 regulated c-Myc and NPM1 ubiquitination. Primary CD34 + CD38- AML blasts were transfected with vector control or HA-Flag-USP36 for 12 h, following by treatment with OST-01 (1 µL/mL) combined with MG132 (10 µM) for 24 h. The cell lysate was immunoprecipitated with anti-c-Myc and anti-NPM1 and immunoblotted with anti-Ubiquitin (Ub) antibodies.