Biological characterization of terpenoids identified by gene expression analyses. (A) Bar chart of FACS analysis indicating significant impairment of viability (single asterisk, P < .01; N = 3 patient samples) of primary CD34⁺ CD38⁻ AML cells versus normal human CD34⁺ CD38⁻ bone marrow treated with 2 μM celastrol. Alongside, a representative FACS analysis of viability is shown. The lower left quadrant (annexin V negative/7-AAD negative) represents viable cells, whereas events in the lower right and upper right panels represent dying and dead cells, respectively. Viability is represented as a percentage of untreated controls. (B) Methylcellulose colony assays of primary human cells treated with 2 μM celastrol. Normal erythroid, myeloid, and AML colony forming units are shown relative to untreated controls. Error bars represent the standard error of the mean. Black bars are normal cells. White bars are AML. Significant selectivity (asterisk) is indicated by either the AML versus myeloid or AML versus erythroid comparisons with celastrol treatment (single asterisk, P < .01; N = 3 patient samples). Viability is represented as a percentage of untreated controls. (C) Bar charts indicate the percent engraftment of AML cells in NOD/SCID mice after 18 hours in culture with 2 μM celastrol (Cel) versus untreated control (Unt). White bars represent the cohort of animals injected with untreated cells; black bars represent 2 μM celastrol treatment. Significant loss of engraftment (P < .01, single asterisk; N = 3 patient samples, 3-4 mice/sample) in NOD/SCID mice is observed with celastrol treatment. No asterisk, P = .11 (4 mice). Overall P value for the treatment is P = .0003. (D) Bar chart indicating viability of phenotypically defined AML cells 24 hours after treatment with 20 μM gedunin (N = 3 patient samples). Viability is represented as a percentage of untreated controls. (E) Representative confocal microscopy images of primary CD34⁺ AML cells treated with 2 μM celastrol, 20 μM gedunin, or left untreated. Top panels show overlays labeling for NF-κB p65 (green) and nucleus (red). Bottom panels show overlays for Nrf2 (yellow) and nucleus (blue). (F) Top panel: EMSAs indicating NF-κB binding relative to untreated (UNT) cells with either 2 μM celastrol (CEL) or 20 μM gedunin (GED) 6 hours after treatment of CD34⁺ AML cells. Bottom panel: immunoblots indicating phospho-p65, HO-1, and β-actin levels at 6 hours in primary CD34⁺ AML treated with 2 μM celastrol (CEL) treatment, 20 μM gedunin (GED), or left untreated (UNT).