Nature Medicine: doi: /nm.3776

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Cameron Campbell
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Stalla and Marcelo Paez Pereda Supplementary Figure 1 Supplementary Figure 1: Quantitative determination of Hsp90-α and Hsp90-β.

1 C terminal Hsp90 inhibitors restore glucocorticoid sensitivity and relieve a mouse allograft model of Cushing s disease Mathias Riebold, Christian Kozany, Lee Freiburger, Michael Sattler, Michael Buchfelder, Felix Hausch, Günter K. Stalla and Marcelo Paez Pereda Supplementary Figure 1 Supplementary Figure 1: Quantitative determination of Hsp90-α and Hsp90-β. Quantification of (a) Hsp90-α and (b) Hsp90-β expression relative to β-actin from Figure 1b (AU arbitrary units; NP normal human pituitary; CPA corticotroph pituitary adenoma; NFPA non functioning pituitary adenoma).

2 Supplementary Figure 2: Supplementary Figure 2: Hsp90-α and Hsp90-β build complexes with GR in AtT 20 cells. Immunoblot of Hsp90-α and Hsp90-β after immunoprecipitation of GR (NA no antibody control; NI non immune IgG; IP immunoprecipitation).

3 Supplementary Figure 3: Supplementary Figure 3: Differential effects of Hsp90 inhibitors on cell cycle distribution in AtT 20 cells. We treated AtT 20 cells with 17 AAG ( µm), novobiocin ( µm), or silibinin ( µm) for 24 h. Cell cycle distribution was determined by propidium iodide staining and FACS analysis.

4 Supplementary Figure 4: Supplementary Figure 4: Specific and Hsp90 dependent 3 H dexamethasone binding to GR. (a) AtT 20 cells were treated with silibinin (30 µm), followed by incubation with 5 nm 3 H dexamethasone ( 3 H Dex) with or without the indicated concentrations of unlabelled dexamethasone (Dex) or RU Shown are means ± s.d. of residual binding of 3 H Dex to GR in AtT 20 cells in the presence of competitor in relation to 3 H Dex alone (**P 0.01). The inset shows GR protein levels. (b) AtT 20 cells were treated for 15 min with 17 AAG (10 µm), followed by incubation with 5 nm 3 H Dex with or without a 500 fold excess of unlabelled Dex. Shown are means ± s.d. of specific binding (total unspecific binding) of 3 H Dex to GR in AtT 20 cells (**P 0.01, control vs. 17 AAG). The inset shows GR protein levels.

The cells were treated with silibinin, and dexamethasone (Dex) or Dex plus RU 38486. Shown are means ± s.d. (*P 0.05; **P 0.

5 Supplementary Figure 5 Supplementary Figure 5: The effects of silibinin require GR. (a) Transient transfection of AtT 20 cells with MMTV Luc. The cells were treated with silibinin, and dexamethasone (Dex) or Dex plus RU Shown are means ± s.d. (*P 0.05; **P 0.01, silibinin plus Dex vs. Dex alone). (b) Transient transfection of AtT 20 cells with TK Luc ( ) or GRE 2 TK Luc ( ). The cells were treated with silibinin and Dex. Shown are means ± s.d. of fold change to control cells (*P 0.05, silibinin plus Dex vs. Dex alone).

6 Supplementary Figure 6: Supplementary Figure 6: 17 AAG reduces the dexamethasone mediated suppression of ACTH. Inhibition of ACTH secretion by AtT 20 cells in the presence of a saturating concentration of dexamethasone (Dex: 100 nm) alone set to 100% and in combination with increasing concentrations of 17 AAG as measured by RIA (*P 0.05; **P 0.01, dexamethasone alone vs. dexamethasone plus 17 AAG).

7 Supplementary methods Quantification of Hsp90 expression. The immunoblots were quantified for Hsp90-α, Hsp90-β, and β-actin using ImageJ. FACS analysis. We seeded AtT 20 cells at 3 x 10 5 cells per well in 6 well plates and left them to attach for 24 h. After that, we changed the medium to cell culture medium with 2% FCS with the indicated drugs. The DMSO concentration was maintained constant for all conditions at 0.2% (vol/vol). After 24 h incubation, we collected the cell supernatant and adherent cells were washed in PBS and trypsinized, pelleted, and fixed in ice cold ethanol (75% v/v final concentration) over night at 20 C. We redissolved the pellet in PBS with propidium iodide (10 µg ml 1 ) and RNAse A (1 µg ml 1 ) for 1 h at room temperature, followed by FACS in a Beckman Coulter EPICS XL. Co immunoprecipitation. We captured GR from AtT 20 lysate as described in Materials & Methods of the main text. Hsp90-α was detected with ADI SPA 840 (1:2,000) and Hsp90-β was detected with Clone E296 (1:2,000). 3 H dexamethasone binding. We cultured and treated AtT 20 cells as described in Materials & Methods of the main text, except that the indicated concentrations of competitor (dexamethasone or RU (Tocris)) were added during incubation with 3 H dexamethasone (4 h at 4 C), or that we incubated AtT 20 cells with 10 µm 17 AAG for 15 min prior incubation with 3 H dexamethasone (4 h at 4 C) in the presence or absence of a 500 fold excess of unlabelled dexamethasone. Reporter assay. We cultured, transfected and subsequently treated AtT 20 cells as described in Materials & Methods of the main text, except that dexamethasone and RU were added for the last 6 h, or that we used as reporters 0.3 µg TK Luc or 0.3 µg GRE 2 TK Luc where indicated with incubation of dexamethasone for the last 18h. ACTH RIA. We cultured and treated AtT 20 cells as described in Materials & Methods of the main text.

FSC-W FSC-H CD4 CD62-L

FSC-W FSC-H CD4 CD62-L Supplementary Fig. 1 a SSC-A FSC-A FSC-W FSC-H SSC-W SSC-H CD4 CD62-L b SSC-A FSC-A FSC-W FSC-A FSC-A 7-AAD FSC-A CD4 IL-9 CD4 c SSC-A FSC-A FSC-W FSC-H SSC-W SSC-H 7-AAD KI67 Annexin-V 7-AAD d I L -5