PU-H71 inhibited the 72 h Alamar blue growth readout in Ewing sarcoma cells. Figure-labeled IC50 values were 0.223 µM in A673 and 0.187 µM in SK-PN-DW cells (three experiments, each in triplicate). Benign-cell comparators showed lower sensitivity in the same assay. These metabolic growth endpoints do not by themselves quantify apoptosis or establish a general normal-tissue safety margin. [2]
Cellular competition for fluorescent PU-H71 probe binding
Cell-based study · K562 cells; primary CML and AML blasts; normal blood-cell comparators [1]
Unconjugated PU-H71 competed with PU-FITC binding more potently in the leukemic models than in normal blood cells. These are apparent cellular competition endpoints, not purified-enzyme IC50 values.
Literature-Reported Treatment Conditions
- Treatment concentration
- 125 nM, 250 nM, 500 nM, 1 µM and 2 µM shown in Figure 1d
- Exposure duration
- 24 h pretreatment according to Figure 1d caption
- Method
- Flow cytometry of fluorescent PU-FITC binding, with vehicle, unstained and TEG-FITC nonspecific-binding controls
- Vehicle
- DMSO stocks reported in SI; vehicle control shown in Figure 1d
- Reported limits
- Dose-response probe incubation timing is not fully specified in the caption. SI also describes a separate 1 µM, 4 h competitor/1 h probe competition test; that schedule is not substituted for Figure 1d. Final DMSO percentage and independent replicate number for Figure 1d are not specified in its caption.
Methods and source locations
Compound reported in the paper: PU-H71 (Zelavespib); Compound label in the paper: PU-H71
Results apply to the reported research material and model; they do not validate an Adooq lot.
Figure 1d and caption; Results: PU-H71 interacts with a restricted fraction of Hsp90; SI Flow cytometry (S19-S20)
Competition with PU-FITC binding in K562 cells: Flow cytometry; value explicitly stated in Results — Results: PU-H71 interacts with a restricted fraction of Hsp90; Figure 1d
Competition with PU-FITC binding in primary CML blasts: Flow cytometry; value explicitly stated in Results — Results: PU-H71 interacts with a restricted fraction of Hsp90; Figure 1d
Competition with PU-FITC binding in primary AML blasts: Flow cytometry; value explicitly stated in Results — Results: PU-H71 interacts with a restricted fraction of Hsp90; Figure 1d
Competition with PU-FITC binding in normal blood cells: Reported normal-cell comparison; not proof of universal normal-tissue selectivity — Results: PU-H71 interacts with a restricted fraction of Hsp90; Figure 1d and cited Supplementary Figure 1d
Preferential loss of Bcr-Abl versus Abl in K562 cells
Cell-based study · K562 chronic myeloid leukemia cells [1]
At 1 µM for 24 h, PU-H71 reduced Bcr-Abl more strongly than native Abl in a representative immunoblot; Hsp90 was displayed as a comparator. This supports differential client susceptibility, without establishing direct Bcr-Abl inhibition.
Literature-Reported Treatment Conditions
- Treatment concentration
- 1 µM
- Exposure duration
- 24 h
- Method
- Western blot versus vehicle
- Vehicle
- DMSO
- Reported limits
- Final DMSO percentage, replicate number and numerical densitometry for this representative blot are not reported in its caption.
Methods and source locations
Compound reported in the paper: PU-H71 (Zelavespib); Compound label in the paper: PU-H71
Results apply to the reported research material and model; they do not validate an Adooq lot.
Figure 2d and caption; Results: PU-H71 selects for oncoprotein-Hsp90 complex species; SI Reagents and Western Blotting (S14)
Bcr-Abl relative to native Abl protein after treatment: Representative Western blot; no numerical fold reduction inferred — Figure 2d; Results: PU-H71 selects for oncoprotein-Hsp90 complex species
Growth inhibition of Ewing sarcoma cells with benign-cell comparators
Cell-based study · Eight Ewing sarcoma cell lines; HS5, SDBMSC and HBVP benign-cell comparators [2]
A 72 h Alamar blue assay showed concentration-dependent reduction in metabolic growth readout. Figure 1A labels A673 and SK-PN-DW IC50 values as 0.223 and 0.187 µM; the three benign comparators had higher labeled IC50 values. This endpoint is distinct from apoptosis.
Literature-Reported Treatment Conditions
- Treatment concentration
- 0.015–2 µM
- Exposure duration
- 72 h
- Method
- Alamar blue assay normalized to respective controls; n = 3 experiments, each in triplicate
- Reported limits
- Final vehicle composition for the proliferation series is unspecified. Some fitted IC50 labels in benign cells exceed the stated tested range; do not treat them as directly observed half-maximal concentrations.
Methods and source locations
Compound reported in the paper: PU-H71 (Zelavespib); Compound label in the paper: PU-H71
Results apply to the reported research material and model; they do not validate an Adooq lot.
Sections 2.1, 2.2, 2.12 and 3.1; Figure 1A and caption
Reduction of A673 Alamar blue growth readout versus control: 72 h Alamar blue assay; n = 3 experiments, each in triplicate — Figure 1A label and caption; Section 2.2
Reduction of SK-PN-DW Alamar blue growth readout versus control: 72 h Alamar blue assay; n = 3 experiments, each in triplicate — Figure 1A label and caption; Section 2.2
Growth inhibition in benign-cell comparators: Same Figure 1A assay; upper-range extrapolation limits apply — Figure 1A and caption; Section 3.1
