Extended Data Figure 3: PU-H71 and its labelled versions are reliable tools to perturb, identify and measure the expression of the high-molecular-weight, multimeric HSP90 complexes in tumours.
From: The epichaperome is an integrated chaperome network that facilitates tumour survival
a, Correlative analysis between binding of a fluorescently (FITC) labelled PU-H71 (PU-FITC) to the panel of cancer cells shown in Fig. 1a (n = 6) and their apoptotic sensitivity to HSP90 inhibition (Pearson’s r, two-tailed). Each data point represents a cell line. b, MDA-MB-468 (type 1) and ASPC1 (type 2) contain similar levels of HSP90 but only MDA-MB-468 expresses the high-molecular-weight chaperome species (see also Fig. 1a). HSP90α and HSP90β levels were quantified by fluorescence microscopy (n = 50; mean ± s.d.; unpaired t-test, NS). Scale bar, 10 μm. c, Association and dissociation of PU-FITC (a FITC labelled PU-H71) from HSP90 was probed in cell homogenates by fluorescence polarization. Average from technical duplicates is shown on the graph. The experiment was carried out twice with similar results. d, Association and dissociation of PU-H71-bait (a solid-support immobilised PU-H71) from HSP90 was probed in cell homogenates by chemical precipitation followed by analyses of HSP90 in the supernatant and of HSP90 isolated on the solid support. A solid-support containing immobilized PU-H71 and an HSP90-inert molecule (control bait) were incubated with cell homogenates for 2 h. The bait-captured cargo was isolated and analysed by western blot (bait). The HSP90 species in the supernatant were separated and analysed as indicated. For isoelectric focusing, both the gel (for experimental duplicates) and the heat map representations of different exposure times are shown for each experimental condition. HMEC cells are shown for reference. Data were repeated independently twice with representative data shown. e, f, Association and dissociation of PU-H71 from type 1 and 2 tumours, measured in cells. Binding of PU-FITC to live cells was analysed by flow cytometry and fluorescence microscopy, as indicated. PU-FITC (1 μM for flow and 5 μM for microscopy) was added to cells and incubated for 4 h before fluorescence signal detection. To show specificity of binding, the signal was competed off in a dose-dependent manner with unlabelled PU-H71. Control FITC, a triethylene glycol labelled FITC. e, Mean from two technical replicates; f, mean ± s.d., n = 50 individual cells, unpaired t-test, ****P < 0.0001. The fluorescence intensity of PU-FITC staining was quantified by ImageJ. Scale bar, 10 μm. g, Association and dissociation of PU-H71 from type 1 and 2 tumours, measured in vivo. Biodistribution of 124I-PU-H71 (a 124I radiolabelled version of PU-H71) was monitored live in tumour-bearing mice. Each mouse bears one xenografted MDA-MB-468 and one ASPC1 tumour, of similar volume, as indicated. Following intraveneous (iv) injection of a tracer amount of the 124I-PU-H71 agent, mice were monitored by micro-positron emission tomography (PET) imaging. Representative images taken at the indicated times post-injection are shown. Note that immediately after injection (1 h timepoint image), the agent is widely and uniformly distributed throughout the body and in each tumour. The off rate from type 1 tumours is slower compared to type 2 and non-transformed tissues (that is, distinct koff from type 1 tumours versus type 2 tumours). The image is representative of five individual mice. In an independent experiment, radioactivity was measured in a gamma-counter upon mouse euthanasia and data were graphed to monitor the time-dependent distribution of PU-H71. Graph; radioactivity, measured as %IDg; injected dose per gram, versus time upon euthanasia (mean ± s.d., n = 8, ASPC1; n = 34, MDA-MB-468, pooled experiments of mice bearing individual tumours). Means were compared by unpaired t-tests between MDA-MB-468 and ASPC1 at each time point (NS, not significant; ***P < 0.001; ****P < 0.0001). h, Same as in g for a therapeutic dose of injected PU-H71, as indicated. Levels of intact PU-H71 in the indicated tumours, tissues and plasma were determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) in mice (n = 5) euthanized at the indicated times post-PU-H71 injection. Graph; mean ± s.d., unpaired t-tests between MDA-MB-468 and ASPC1 (NS, not significant; ***P < 0.001). i, Dose- and time-dependent binding of PU-H71 and H9010 (an anti-HSP90 antibody) to HSP90 species expressed in type 1 and type 2 tumour cells. C, control beads containing an HSP90-inert molecule. PU, 10 μl PU-H71 wet beads; 2×PU, 20 μl PU-H71 wet beads; H9010; 2 μl antibody immobilized on agarose beads. Because the IgG interferes with the HSP90 signal (see the high molecular smear in the native gels), native lysates were used for a control (input). Graph shows quantification of time-dependent changes in HSP90 species. j, Representative fluorescence microscopy images of live cells stained with PU-FITC (top) as compared to antibodies specific for HSP90 (bottom). rbtIgG, rabbit IgG control, msIgG, mouse IgG control. Scale bar, 10 μm. Micrograph is representative of four captured images. For uncropped gel data, see Supplementary Fig. 1.
