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Figure 1 | Journal of Hematology & Oncology

Figure 1

From: Heat shock factor 1 is a potent therapeutic target for enhancing the efficacy of treatments for multiple myeloma with adverse prognosis

Figure 1

HSP90 and HSF1 inhibitors are potent antimyeloma drugs. (A) HSP expression was determined in a panel of HMCLs belonging to CD-1/2, MS, and MF groups ([5] and Additional file 2) by Western blotting. Blots were incubated with the following Abs: anti-HSP27, −HSP70, and -HSP90 from Santa Cruz Biotech.; anti-GAPDH from Life Technologies; and α-tubulin from Dako. Abs anti-GAPDH and -α-tubulin served for gel loading control. (B) Affymetrix gene expression profiles of purified myeloma cells (Additional file 1). HSP90AA1, HSPB1, and HSPA4 gene expressions (in Affymetrix signal units) are indicated for each patient in the different molecular groups according to [6]: HY, CD-1/2, LB corresponding to standard risk in light gray, MS, or MF, and PR corresponding to high risk in dark gray. The expression of those genes was also analyzed in normal bone marrow plasma cells (BMPC). p < 0.001 and NS (not significant) with Student’s t test. (C) The cell lines used in this assay are described in Additional file 1. Cells (0.5 × 106/ml) were seeded for 48 h in 96-well plates and treated with serial dilutions of 17-AAG (20 to 0.3 μM) or KNK-347 (200 to 3.1 μM). Cell death was then assessed using flow cytometry with the combined analysis of APO2.7 (Beckman Coulter) staining according to the manufacturer’s recommendation and the altered cellular morphology characteristics of apoptosis (lower FSC-H and higher SSC-H). Flow cytometry analysis was performed on a FACSCalibur using the CellQuest software (BD Biosciences). The LD50 was defined as the concentration that killed 50% of cells (mean of 3 experiments). (D) L363 cells were treated for 24 h with 100 μM KNK-437 or 5 μM 17-AAG. Western blots were obtained as before. Ab anti-MCL1 was obtained from Santa Cruz Biotech. and anti-BCL2 from Dako (Glostrup, Denmark). The cleaved forms of PARP and procaspase 3 are arrowed. ▲ marked a non-specific band.

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