EMBO/soda-nel huggingface dataset:
The Hippo pathway drives the cellular response to hydrostatic pressure (2022)
Figure 1

Figure 1. Hydrostatic pressure activates YAP/TAZ.

A PhosTag-based Western blot probed for YAP reveals increased levels of unphosphorylated (downshift) YAP in HEK293A cells in response to cyclic 0.1Hz 200mbar hydrostatic pressure (+) compared to control (-).

B PhosTag-based Western blot probed for TAZ reveals increased levels of unphosphorylated (downshift) TAZ in HEK293A cells in response to cyclic 0.1Hz 200mbar hydrostatic pressure (+) compared to control (-).

C Western blots from cell lysates as in (A) and (B) show total protein levels in control (-) and in cell lysates obtained from cells exposed to hydrostatic pressure (+) stimulation in HEK293A cells. Note the decrease in the inhibitory p127 YAP signal as well as increase of YAP/TAZ targets CTGF and CYR61 levels upon hydrostatic pressure.

D PhosTag-based Western blot probed for YAP reveals increased levels of unphosphorylated (downshift) YAP in 143B cells in response to hydrostatic pressure (+) compared to control (-).

E Western blot shows total protein levels in control (-) and with hydrostatic pressure (+) stimulation in 143B cells from cell lysates as in D.

F HEK293A cells at steady state (upper) or upon cyclic 0.1Hz 200mbar hydrostatic pressure (lower) were analysed by immunofluorescence. Cells are labelled for Hoechst (blue) and YAP (green). Scale bar = 20µm. Graph on the right depict quantification of nuclear-to-cytoplasmic (Nucl/Cyt) ratio of YAP in cells as shown on the left. Each dot represents a single cell and data are pooled from three independent experiments. Error bars represent mean ± 95% CI. Mann-Whitney U test. ***p < 0.001.

G 143B cells at steady state (upper) or upon cyclic 0.1Hz 200mbar hydrostatic pressure (lower). Graph on the right show quantification of nuclear-to-cytoplasmic (Nucl/Cyt) ratio of YAP in 143B from images as those shown on the left. Each dot represents a single cell and data are pooled from three independent experiments. Error bars represent mean ± 95% CI. Mann-Whitney U test. ***p < 0.001.

H Western blot confirming no YAP and TAZ expression in two independent (#1 and #2) YAP/TAZ double knockout (Y/T DKO) clones.

I Relative expression levels of YAP/TAZ target genes CYR61 and CTGF in HEK293A WT and Y/T DKO clones in response to cyclic 0.1Hz 200mbar hydrostatic pressure. Data from six independent experiments Error bars represent mean ± SD. Kruskal-Wallis test with Dunn's post hoc. **p=0.0661 (WT CYR61), ***p= 0.0001 (WT CTGF), p=0.3924 (Y/T DKO#1 CYR61), p=0.4616 (Y/T DKO#1 CTGF), p>0.999 (Y/T DKO#2 CYR61), p=0.0782 (Y/T DKO#2 CTGF).

J CYR61 and CTGF gene expression levels induced by hydrostatic pressure in (I) of Y/T DKO #1 and #2 normalised to WT. Kruskal-Wallis test with Dunn's post hoc. *p=0.0259 (Y/T DKO#1 CYR61), **p=0.0007 (Y/T DKO#1 CTGF), *p=0.0155 (Y/T DKO#2 CYR61), ***p<0.001 (Y/T DKO#2 CTGF).
The Hippo pathway drives the cellular response to hydrostatic pressure (2022)
Figure 3

Figure 3. Cellular response to hydrostatic pressure is regulated by the actin cytoskeleton.

A Cell volume measured by DHM of HEK293A cells treated with 0.5µM (left) and 2µM (right) Cytochalasin D. Each dot represents a single cell and error bars represent mean ± 95% CI. Data pooled from four independent experiments. Mann-Whitney U test. **p=0.0033 (WT 0.5µM), ***p<0.001 (Y/T DKO 0.5µM), ***p=0.004(LATS1/2 DKO 0.5µM), ***p<0.001 (WT 2µM), **p=0.0022 (Y/T DKO 2µM), *p=0.0145 (LATS1/2 DKO 2µM).

B Fold change in cell volume upon treatment with 0.5µM (left) and 2µM (right) Cytochalasin D relative to untreated control. Each dot represents a single cell and error bars represent mean ± 95% CI. Data pooled from four independent experiments. Mann-Whitney U test. p=0.1197 (WT vs Y/T DKO), p>0.9999 (all other comparisons).

C Representative immunofluorescence images showing Phalloidin-labelled actin (red) and Hoechst (blue) in Latrunculin B (LatB) (0.5µM) or Cytochalasin D (CytD) (2µM)-treated HEK293A cells compared to control across Hippo pathway genome-edited cells as shown. Scale bar = 20μm.

D Average change in cell volume obtained by DHM in response to hydrostatic pressure with 2µM Cytochalasin D (CytD) treatment in WT, Y/T DKO and LATS1/2 DKO cells. Each dot represents a single cell and error bars represent mean ± 95% CI. Data pooled from four independent experiments. Kruskal-Wallis test with Dunn's post hoc. **p=0.0044 (WT con vs CytD), p*=0.0409 (Y/T DKO con vs CytD), p>0.9999 (LATS1/2 DKO con vs CytD), ***p=0.0003 (WT con vs Y/T DKO con), ***p<0.001 (WT con vs Y/T DKO CytD), ***p<0.001 (Y/T DKO CytD vs LATS1/2 DKO CytD).

E Fold difference in average change in cell volume in response to dynamic hydrostatic pressure from (H) normalised against untreated cells. Each dot represents a single cell and error bars are 95% CI. Mann-Whitney U test. **p=0.0069 (WT vs. Y/T DKO), **p=0.0031 (WT vs LATS1/2 DKO).

F Average change in cell volume in response to hydrostatic pressure with 0.5µM Latrunculin B (Lat B) treatment in WT, Y/T DKO and LATS1/2 DKO cells. Each dot represents a single cell and error bars represent mean ± 95% CI. Data pooled from four independent experiments. Kruskal-Wallis test with Dunn's post hoc. ***p<0.001 (all comparisons), p=0.0972 (LATS/12 DKO con vs LatB), *p=0.0216 (Y/T DKO LatB vs LATS1/2 DKO LatB).

G Fold difference in average change in cell volume in response to cyclic 0.1Hz 100mbar hydrostatic pressure from (F) normalised against control. Each dot represents a single cell and error bars represent mean ± 95% CI. Mann-Whitney U test. p=0.1472 (WT vs. Y/T DKO), **p=0.0015 (WT vs LATS1/2 DKO).
The Hippo pathway drives the cellular response to hydrostatic pressure (2022)
Figure 5

Figure 5. Hydrostatic pressure promotes clathrin-dependent internalisation.

A Confocal IF images labelled with Hoechst (Blue) and internalised Transferrin (Trf). Cells were allowed to internalize for ten minutes, fixed and acid stripped to remove surface bound Trf. Hydrostatic pressure (H.P.) promotes transferrin uptake in WT and LATS1/2 DKO cells. Scale bar = 20µm.

B Quantification of transferrin uptake in WT, Y/T DKO and LATS1/2 DKO cells treated with hydrostatic pressure compared to steady state. Each dot represents a single cell and error bars represent mean ± 95% CI. Data from four independent experiments. Kruskal-Wallis test with Dunn's post hoc. Steady-state statistical analysis is shown in blue, transferrin uptake induced by pressure is shown in black. ***p<0.001 (WT con vs +H.P.), p=0.2375 (Y/T DKO con vs +H.P.), ***p<0.001 (LATS1/2 DKO con vs +H.P.), p=0.2764 (WT con vs Y/T DKO con), p=0.0808 (WT con vs LATS1/2 DKO con), ***p=0.0004 (Y/T DKO con vs LATS1/2 DKO con).

C Confocal IF images myc tagged AP180C (green) cells showing internalised Trf (red) and stained with Hoechst (blue). AP180C inhibits transferrin uptake in WT and Y/T DKO cells. Scale bar = 20µm.

D Quantification of transferrin uptake in AP180C positive cells compared to vector control from images as in C. Each dot represents a single cell from four independent experiments and error bars represent mean ± 95% CI. Mann-Whitney U test. ***p < 0.001 (for both comparisons).

E Confocal IF images of HEK293A WT cells showing subcellular localisation of YAP (red) in AP180C (green) negative and positive WT and LATS1/2 DKO cells. Cells also stained with Hoechst (blue).

F Quantification of cytoplasmic-to-nuclear ratio of YAP in AP180C positive WT and LATS1/2 DKO cells compared to vector control from images as in E. Each dot represents a single cell pooled from four independent experiments. Error bars represent mean ± 95% CI. Mann-Whitney U test. ***p=0.0008 (WT), p=0.9318 (LATS1/2 DKO).

G Cell response to hydrostatic pressure with AP180C-mediated inhibition of clathrin-dependent endocytosis. Each dot represents a single cell pooled from four independent experiments. Error bars represent mean ± 95% CI. Data pooled from four independent experiments. Kruskal-Wallis test with Dunn's post hoc. ***p=0.0005 (WT), p=0.3930 (Y/T DKO), p=0.2929 (LATS1/2 DKO).
Chemokines and galectins form heterodimers to modulate inflammation (2020)
Figure 1

Figure 1. Physical interaction of Gal-3 and Gal-1 with CC and CXC chemokines.

A-E Chemokine-galectin interactions were detected by using a solid-phase immunoassay. For this, 46 human CC and CXC chemokines were adsorbed on nitrocellulose membranes and the stripes incubated in parallel with (A) TBS or (B) TBS containing biotinylated galectins (the representative image shows a processed membrane tested with labeled Gal-3). Signals had been generated by using SA-HRP and chemiluminescence reagents. (C-D) The blots were subjected to densitometric analysis, and (E) all independent experiments were combined (binding chemokines in light blue, Gal-3: n = 5, Gal-3 CRD: n = 5, Gal-1 n = 4).

F-H For SPR-based experiments, (F) Gal-3 (density 650 RU), (G) Gal-3 CRD (density 1180 RU), and (H) Gal-1 (density 130 RU) were immobilized and increasing concentrations of CXCL12 were passed over the flow cells. The red curve represents a single-site fit to the data. Insets are representative sensorgrams of CXCL12 testing on immobilized galectin. Data represent the mean ± SD from six (F) or three (G and H) independent experiments.
Chemokines and galectins form heterodimers to modulate inflammation (2020)
Figure 2

Figure 2. Formation of the CXCL12/Gal-3 CRD heterodimer.

a-b Expansions of 1H-15N HSQC spectra are overlaid and Δδ values are plotted vs. the amino acid sequences, along with secondary structures: (a) 10 µM 15N-enriched CXCL12 alone (black peaks) and in the presence of 330 µM Gal-3 CRD (red peaks; assignments reported by Murphy et al.[74]) and (b) 30 µM 15N-enriched Gal-3 CRD alone (black peaks) and in the presence of 500 µM CXCL12 (red peaks; assignments for Gal-3 reported by Ippel et al. [34]).

c-e X-Ray crystal structures are depicted (c) for the CXCL12 homodimer (PDB access code 4UAI; monomer in magenta) and (d) for Gal-3 CRD bound with lactose (PDB access code 1A3K; in light green). (e) The energy-minimized structure of the CXCL12/Gal-3 CRD heterodimer was calculated by MD simulations. Residues with the largest Δδ values in HSQC spectra are highlighted in red (2SD-1SD) and orange (1SD-mean).

F A model structure of the CXCL12/Gal-3 CRD heterodimer interface is shown with Gal-3 CRD in light green, the CXCL12 monomer in magenta, mutated residues in red, and hydrogen bonds as dashed orange lines.
Chemokines and galectins form heterodimers to modulate inflammation (2020)
Figure 3

Figure 3. Gal-3-mediated inhibition of CXCL12-induced leukocyte migration in vitro.

a-c Jurkat T cells migrated in the presence of 10 nM CXCL12 with increasing concentrations of (a) Gal-3, (b) Gal-3 CRD, and (c) Gal-1 (A-C: n = 4).

D Human CD4+ T cells migrated in the presence of 10 nM CXCL12 alone and with 10 nM Gal-3 (dark green), Gal-3 CRD (light green), and Gal-1 (blue, n = 3).

E-F (E) Jurkat T cells migrated to increasing concentrations of CXCL12 alone (black) and CXCL12 in the presence of 0.1 nM of Gal-3 CRD (light green). (F) The inhibitory effect of Gal-3 CRD is shown as percentage of the chemotactic effect of CXCL12 (E-F: n = 3).

G Jurkat T cells were allowed to migrate in the presence of CXCL12 at increasing concentrations alone and with 0.1 nM Gal-1 or 100 nM Gal-1 (n=3).

H Jurkat T cells did not migrate in the sole presence of 1 nM to 1 µM Gal-3, Gal-3 CRD (both n = 5), or Gal-1 (n = 4).

I The viability of Jurkat T cells was assessed after incubation with 1 µM CXCL12 alone and in the presence of Gal-3, Gal-3 CRD and Gal-1 (n = 2).

J-K (J) THP-1 cells (K) and neutrophils migrated in the presence of 10 nM CXCL12 alone and with 10 nM Gal-3, Gal-3 CRD and Gal-1 (J-K: n = 3).

L Human eosinophils migrated in the presence of 10 nM CCL26 alone and with 10 nM Gal-3, Gal-3 CRD, and Gal-1 (n = 3).

M CD4+ T cells migrated in the presence of 1 nM CCL17 alone and with equimolar concentrations of Gal-3, Gal-3 CRD, and Gal-1 (n = 4).

Data information: Cell migration is shown as absolute cell counts. Data represent the mean ± SD from the indicated number of independent experiments each performed in three technical replicates, and these were statistically analyzed by using (A-C,F-G,J) a single sample t-test or (D,I,J-M) by an unpaired t-test against the effect of the chemokine alone or as indicated (*= p ≤ 0.05, **= p ≤ 0.01, ***= p ≤ 0.001).
Chemokines and galectins form heterodimers to modulate inflammation (2020)
Figure 4

Figure 4. Peritoneal recruitment of leukocytes by CXCL12 in the presence of Gal-3

A-B The peritoneal recruitment of (A) CD45+/CD115-/Ly6G+ neutrophils and (B) CD45+/CD115+/Ly6Chi classical monocytes in C57BL/6J mice was assessed 4 hours after intraperitoneal (IP) injection of 500 nM CXCL12 alone (black) and in the presence of 50 nM Gal-3 (dark green; A-B: n = 7 mice).

C The peritoneal recruitment of classical monocytes after IP injection of PBS (n = 6) or TG in C57BL/6J WT (dark green, n = 10 mice) and Gal-3-/- (black, n = 5 mice) mice was assessed after 18 h. Where indicated, the mice received an IP injection of CXCR4 antagonist AMD 3465 12 hours prior to the experiment.

D The concentration of CXCL12 concentration was determined by ELISA on the peritoneal lavage normalized with levels from the mesenterium (n = 4 mice).

E CXCR4 expression levels on Ly6Chi monocytes of the blood and the peritoneal lavage after 18 h of TG stimulation were determined by flow cytometry and indicated as mean fluorescence intensity (MFI) (n = 4 mice).

Data information: Cell migration to the peritoneum is shown as cells/mL lavage. Data represent the mean ± SD from the indicated number of mice and were statistically analyzed by using the unpaired t-test, as indicated (*= p ≤ 0.05, **=p ≤ 0.01).
Chemokines and galectins form heterodimers to modulate inflammation (2020)
Figure 5

Figure 5. Inhibition of CXCL12-induced leukocyte migration by Gal-3 mutants and the role of galectin/glycan interactions in vitro.

A-B Jurkat T cells migrated in the presence of 10 nM CXCL12 with increasing concentrations of E185A (orange, n = 4) and N222A (red, n = 5) mutants of (A) Gal-3 and (B) Gal-3 CRD.

C-D Jurkat T cells migrated in the presence of 10 nM CXCL12 with 10 nM (C) Gal-3 and (D) Gal-3 CRD mutants as indicated (C-D: n = 3).

E Aggregation of Jurkat T cells in the presence of 1 nM to 1 µM Gal-3 and Gal-1 alone and with 70 mM lactose, and Gal-3 CRD, was determined by flow cytometry (n = 4).

F-J Jurkat T cells migrated with 10 nM CXCL12 alone and in the presence of (F) 1 µM Gal-3, (G) Gal-1, (H) Gal-3 CRD and (I) 0.1 nM Gal-3 and (J) Gal-3 CRD in the presence of 70 mM lactose or cellobiose or after treatment of cells with 150 µM DMJ.

Data information: Cell migration is shown as absolute cell counts. Data represent the mean ± SD from the indicated number of independent experiments each performed in three technical replicates, and these were statistically analyzed by using (A-B,E) a single sample t-test or (C-D,F-J) by an unpaired t-test against the effect of the chemokine alone or as indicated (*= p ≤ 0.05, **= p ≤ 0.01, ***= p ≤ 0.001).
Chemokines and galectins form heterodimers to modulate inflammation (2020)
Figure 6

Figure 6. Inhibition of CXCL12-induced CXCR4 signaling by Gal-3 CRD.

a HEK cells transfected with a luminescent cAMP sensor were incubated with 10 nM CXCL12 alone and in the presence of 10 nM Gal-3 CRD followed by stimulation with forskolin, an activator of adenylate cyclase. Results are shown as luminescence relative to baseline (RLU, example of n = 3).

b The effect of 10 nM Gal-3 CRD upon stimulation with 10 nM CXCL12 prior to forskolin stimulation, was tested. Control experiments were performed as indicated (n = 3, four technical replicates).

c HEK cells transfected with a RlucII-conjugated CXCR4 and an eYFP-β-arrestin 2 construct were stimulated with 10 nM CXCL12 alone and in the presence of 10 nM Gal-3 CRD. Control experiments were performed as indicated. Results are given as the net BRET ratio (i.e. ratio of emissions at 535/485 nm minus the ratio of mock cells, n = 5, three technical replicates).

D Internalization of CXCR4 with 10 nM CXCL12 alone and in the presence of 10 nM Gal-3 CRD was assessed by incubation with an APC-conjugated anti-CXCR4 antibody (baseline signal = 100%, n = 8, two technical replicates).

E 100 nM CXCL12 and Gal-3 were added to Jurkat T cells, and their co-localization was assessed on the cell surface by PLA. Control experiments were performed as indicated (representative example of n = 3). White scale bar: 10 µm.

F-G Jurkat T cells were incubated with 100 nM of Gal-3 CRD Alexa Fluor 555 alone, with 10 µM AMD 3100, with 100 nM CXCL12 or with CXCL12 and AMD 3100. Experiments were performed (f) without and (g) in the presence of 70 mM lactose (F-G: n = 3, two technical replicates).

H Jurkat T cells were incubated with 100 nM CXCL12 FITC alone and with increasing concentrations of Gal-3 CRD as indicated (n = 4, two technical replicates).

Data information: Data represent the mean ± SD of the indicated number of independent experiments and were statistically analyzed by (F-g) unpaired t-test or (b-D,h) single sample t-test against the effect of the chemokine alone or as indicated (*= p ≤ 0.05, **= p ≤ 0.01).

Expanded View Figure legends
Mesenchymal tumor organoid models recapitulate rhabdomyosarcoma subtypes (2022)
Figure 2

Fig. 2 - Early detection of tumor cells during culturing, retained marker protein expression and heterogeneity in gene expression:

(A) RT-qPCR of early passage RMS tumoroid models shows positivity for at least one gene used in standard-of-care pathology analysis (DES, MYOG, or MYOD1). Conventional RMS cell lines (RD and RH30) were used as positive controls, while two Synovial Sarcoma (SS000DAZ and SS077) tumoroid models were used as negative controls. Gene expression was normalized to the expression of a house-keeping gene and human reference RNA (HREF) via the ΔΔCq method. Each tumoroid line was measured once with four technical replicates with the error bars representing the standard deviation of said technical replicates.

(B) RT-qPCR of early passage RMS tumoroid models reliably detects the aberrant fusion transcripts. Fusion gene expression was normalized to the expression of a house-keeping gene via the ΔCq method. Each tumoroid line was measured once with four technical replicates with the error bars representing the standard deviation of said technical replicates.

(C) Morphological (via H&E) and immunohistochemical (IHC) comparison of RMS tumors and derived RMS tumoroid models shows retained marker protein (Desmin, Myogenin and MYOD1) expression and cellular morphology. Scale bars equal 200 µm (RMS012, RMS102) or 100 µm (RMS000HQC).

(D) t-SNE projection of single-cell transcriptomes from the RMS127 and RMS444 tumoroid models. Plots on the right show the normalized expression values, per single cell, of MYOG, MYOD1, DES and MKI67, respectively.
Mesenchymal tumor organoid models recapitulate rhabdomyosarcoma subtypes (2022)
Figure 6

Fig. 6 - RMS tumoroid models can be molecularly edited using CRISPR/Cas9 with P53 deficient eRMS being more sensitive to the checkpoint kinase inhibitor prexasertib:

(A) Transfection and selection strategy to achieve TP53 knockout in a TP53 wildtype tumoroid model.

(B) Western Blot analysis of TP53 wildtype (WT) and knockout (KO) RMS tumoroid line RMS012. Histone 3 (H3) served as loading control.

(C) Dose-response curve of TP53 WT and KO cells treated with the Chk-1 inhibitor prexasertib. Thin lines with numbers indicate individual biological replicates (n = 3) while thick lines indicate fitted lines over all replicates. The statistical significance of the differences in fitted IC50 values between WT and KO were obtained using a two-sided t-test (p = 0.008).

Table legends

Table 1: Overview of available RMS tumoroid models in the collection. Patients are numbered to allow identification of RMS tumoroid models derived from the same patient. For additional clinical parameters see Dataset EV1.

Expanded view figure legends
A clinically-compatible drug-screening platform to prioritize anti-metastasis compounds (2022)
Figure 2

Figure 2 - METPlatform is compatible with the evaluation of the metastasis-associated microenvironment.

A Schema of the experimental design.

B Representative images of organotypic cultures with established metastases with various glial components of the microenvironment labelled. Scale bar: 75 µm. Each individual condition was evaluated in several organotypic cultures (3-6 slices).

C Quantification of the bioluminescence signal emitted by established H2030-BrM brain metastases in each organotypic culture at day 3 normalized by their initial value at day 0 (before the addition of DMSO or any compound). The final value in the graph is normalized to the organotypic cultures treated with DMSO. Values are shown in box-and-whisker plots where the line in the box corresponds to the mean. The boxes go from the upper to the lower quartiles and the whiskers go from the minimum to the maximum value (n=5-6 organotypic cultures, 1 independent experiment). P value was calculated using two-tailed t-test.

D Quantification of LDH levels in the conditioned media of organotypic slices cultured during 3 days relative to a lysate of the same preparation. Values are shown as mean + s.e.m. (n=3 organotypic cultures per experimental condition, 1 independent experiment).
A clinically-compatible drug-screening platform to prioritize anti-metastasis compounds (2022)
Figure 3

Figure 3 - Brain metastases are positive for HSP90.

A-C Immunofluorescence against HSP90 in mouse brains with established metastases. (A) HSP90 positive structures in areas not affected by the metastasis includes the medial habenula, where neurons co-localize with the chaperone. Scale bars: 100 µm (low magnification), 50 µm (medial habenula nucleus), 12 µm (high magnification neurons). (B) Established metastases from different primary origins and oncogenomic profiles stained with HSP90. Dotted lines delineate the metastasis (cc: cancer cells). Scale bars: 75 µm. (C) Iba1 colocalizes with HSP90 within areas affected by metastases. BB: bisbenzamide. Scale bar: 75 µm (low magnification), 12 µm (high magnification).

D Immunohistochemistry against HSP90 was performed in human brain metastases (n=60) from lung (40 cases) and breast cancer (20 cases).

E Representative human brain metastases showing different intensities or scores for HSP90. Scale bar: 50 µm.

F Quantification of HSP90 in human brain metastases. 59 out of 60 (98%) showed positive staining of HSP90 in the tumor, 15 (25%) scored with 3 (strong), 36 (60%) with 2 (moderate) and 8 (13%) with 1 (weak) according to the signal intensity of HSP90 in the cytoplasm of cancer cells.

G Human brain metastases (n=30) and their matched primary tumors (n=28 lung and n=2 breast) were evaluated and compared for HSP90 expression by immunohistochemistry.

H Quantification of HSP90 in human primary tumors. 29 out of 30 (97%) showed positive staining of HSP90 in the tumor, 6 (20%) scored with 3 (strong), 10 (34%) with 2 (moderate) and 13 (43%) with 1 (weak) according to the signal intensity of HSP90 in the cytoplasm of cancer cells.

I Schema showing HSP90 scores in matched pairs of primary tumor and brain metastasis. Red: increase of HSP90 score from primary to brain metastasis; green: decrease of HSP90 score; gray: no changes in HSP90 score.

J-K Representative human brain metastases showing different percentages of nuclear HSP90. Scale bars: (J) 50 µm; (K) low magnification: 100 µm; high magnification: 10 µm. Black arrows point to cancer cells positive for HSP90 in the nucleus.

L Quantification of nuclear HSP90 in human brain metastases. 54 out of 60 samples (90%) showed positive nuclear HSP90 in the tumor. 27 (45%) showed 1-5% (moderate) and 27 (45%) showed >5% (high) of nuclear HSP90.

M Quantification of nuclear HSP90 in human primary tumors. 19 out of 30 (63%) showed positive nuclear HSP90 in the tumor. 9 (30%) showed 1-5% (moderate) and 10 (33%) showed >5% (high) of nuclear HSP90.