Compared with HL-60, HL-60/LR cells lack p-HSF1 and hsp70, and LAQ824 treatment did not increase acetylation of hsp90 or induce p21 levels in HL-60/LR cells. (A) HL-60 and HL-60/LR cells were treated with the indicated concentrations of LAQ824 for 8 hours. After this, Western blot analyses were done for hsp70, hsp90, and HSF-1. (B) HL-60 and HL-60/LR cells were treated with the indicated concentrations of LAQ824 for 24 hours. Then total cell lysates were harvested and immunoblot analyses were done for p21, p15, CEBPα, and acetylated α-tubulin. The levels of β-actin expression in the lysates served as the loading control. (C) After treatment with the indicated concentrations of LAQ824, immunoprecipitates of hsp90 were immunoblotted with anti–acetyl lysine or anti-hsp90 antibody. (D) HL-60 and HL-60/LR cells were treated with 250 nM LAQ824 for 8 hours. Then, cell lysates were harvested and immunoblot analyses were done with anti–acetylated lysine 69 hsp90 or anti-hsp90 antibody. The levels of β-actin in the lysates served as the loading control. (E) HL-60 and HL-60/LR cells were treated with 250 nM LAQ824 for 8 hours. Then, cells were cytospun and fixed on slides, and stained with FITC-conjugated anti-acetylated lysine 69 hsp90 antibody (green) and/or DAPI (blue), and imaged with a Zeiss LSM510 confocal microscope with a 63×/1.2 NA water-immersion objective lens.