HSP90 inhibition induces apoptosis of BL cells. (A, top) Heatmap of –log10 (IC50) values for the indicated compounds and BL cell lines. The hierarchical clustering of cell lines based on the corresponding drug response profiles is indicated. (Bottom) Dose-response curves for the HSP90 inhibitor AT13387 in the indicated BL cell lines. (B) Median absolute deviation (log10) of IC50 values vs median IC50 (–log10) across BL cell lines for all tested compounds. Compounds exhibiting an IC50 value <1 μM are labeled. Point sizes are proportional to median absolute deviation values. (C) Anti-HSP90α/β immunoblots of cleared cellular lysates (CCLs) from primary B cells and lymphoma cell lines. Actin was used as loading control. (D) Immunohistochemical staining of lymph node tissues from patients with BL, ABC and germinal center B-cell-like (GCB) DLBCL, gray-zone lymphoma, or healthy donors (GC, germinal center; MZ, marginal zone) with anti-HSP90α and anti-HSP90β antibodies. (Left) Representative images (40-fold magnification) are shown. (Right) All tissue sections were evaluated by 2 independent pathologists using a 3-stage staining score according to the signal intensity. (E) IC50 values for AT13387 and STA-9090 inhibitors after 48 hours of HSP90 inhibition determined by an Annexin V/7-AAD–based flow cytometric apoptosis assay. IC50 values were estimated from Annexin V/7-AAD double negative viable cells. (F) Anti-HSP90 immunoblots of CCL from Daudi cells expressing either control shRNAs, HSP90-specific shRNA, or HSP90-specific shRNA together with murine HSP90 (rescue), at day 2 after lentiviral transduction. (G) Corresponding Annexin V/7-AAD-based apoptosis readout for cells in panel F. Error bars are mean ± standard deviation (SD), n = 3. P values are from a 2-way analysis of variance test; ns, not significant.