Cell culture and western-blotting experiments
NFκB activation was quantified using a stable NFκB luciferase reporter cell line of HEK293 cells with chromosomal integration of a luciferase reporter construct regulated by 6 copies of the NFκB response element (Panomics, CA, USA). Cells were grown in DMEM containing 10% serum, 1% penicillin/streptomycin/fungizone and 100 μg/ml of hygromycin B. Confluent cells were treated with 20 pg/ml of TNFα (Sigma, MO, USA) to induce NFκB activation and with a dose range of celastrol for 3 hours. Celastrol was purchased by Gaia Chemical Corporation (CT, USA). Luciferase activity was detected with the Luc-Screen Extended-Glow from Applied Biosystem (CA, USA) and a Synergy HT Biotek chemoluminescent reader (VT, USA). The medium surrounding the cells was collected and used to assess cytotoxicity using a Lactate Dehydrogenase (LDH) assay (Roche Diagnostics, Germany) according to the manufacturer's protocol. Similar experiments were also conducted with 100 ng/ml of phorbol 12-myristate 13 acetate (PMA) purchased from Sigma (MO, USA) instead of TNFa to induce NFκB activation (data not shown). HEK293 cells were used to assess the effect of celastrol on the activation of canonical members of the NFkB signaling pathway following PMA stimulation and on BACE-1 expression levels. Briefly, HEK293 cells were treated 100 ng/ml of PMA or a combination of PMA with celastrol (5 mM) for 15 minutes. Celastrol effects on RAF-1, MEK1/2, p44/42 MAPK and IKBa phosphorylation were monitored by western-blots as detailed below.
7 W CHO cells overexpressing wild-type human APP  were grown in DMEM (ATCC, VA, USA) containing 10% fetal bovine serum (Invitrogen, CA, USA), 1% penicillin/streptomycin/fungizone (Cambrex, ME, USA) and 0.3% Geneticin (Invitrogen, CA, USA) and used to determine the impact of celastrol on APP processing and Aβ production. Confluent cells were treated for 24 hours with different doses of celastrol.
Cellular proteins were extracted with 150 μL of ice-cold M-PER Reagent (Pierce Biotechnology, Rockford, IL, USA) containing 1 mM phenylmethanesulfonyl fluoride, 1× of protease cocktail inhibitor (Roche, Inc., USA) and 1 mM sodium orthovanadate. Samples were sonicated, denatured by boiling in Laemmli buffer (Bio-Rad, Hercules, CA, USA) and resolved onto 4-20% gradient polyacrylamide gels (Bio-Rad, Hercules, CA, USA). After electrotransfering onto polyvinylidene difluoride membranes, western-blots were immunoprobed with an anti-APP C-terminal (751-770) antibody (EMD Biosciences Inc., San Diego, CA, USA), with an anti-actin antibody (Chemicon, Temecula, CA, USA) used as a reference antibody to quantify the amount of proteins electrotransferred, with a BACE-1 antibody (Invitrogen, CA, USA), with a phospho-IKBα (ser32) antibody, with a phospho-NFκB p65 (ser 536) antibody, with a phospho-Raf-1 (Ser338) antibody, with a phospho-MEK1/2 and a phospho-p44/42 MAPK antibody using dilutions recommended by the manufacturers. All phospho-specific antibodies were purchased from Cell Signaling Technology Inc (MA, USA). Additionally, sAPPα was detected by Western-blot in the culture medium surrounding 7 W CHO cells using the antibody 6E10 (Signet Laboratories Inc., MA, USA) which recognizes the amino-acids 1-17 of Aβ and sAPPβ was detected in the culture medium using an anti-human sAPPβ antibody (Immuno-Biological Laboratories Co. Ltd., Gunma, Japan). APP CTF/actin, sAPPβ/sAPPα signals, phospho-IKBα/actin, phospho-NFκB p65/actin, phpspho-RAF-1/actin, phospho-MEK/actin, phospho-p44/42 MAPK/actin, BACE-1/actin signal intensity ratios were quantified by chemiluminescence imaging with the ChemiDocTM XRS (Bio-Rad, Hercules, CA, USA). Aβ 1-40 and Aβ 1-42 were quantified in the culture media of 7 W CHO cells using commercially available ELISAs following the manufacturer's recommendations (Invitrogen, CA, USA).
Short hairpin RNAs (shRNAs) were used to stably knock-down the cdc37 gene in HEK293 cells overexpressing APPsw cells. Three different cdc37 shRNAs and a scrambled control shRNA (control cells) were used for transfection. Cdc37 shRNAs cloned into the pRs vector were purchased from Origene (Rockville, MD, USA). Approximately one million HEK/APPsw cells detached with TripLE (Invitrogen, IL, USA) were mixed together with 10 μg of shRNA vectors in Bio-Rad (Hercules, CA, USA) gene pulser cuvettes and electroporated using a square wave protocol (110 V, one 25 ms pulse length using a Bio-Rad gene pulser). Forty-eight hours after, the culture media were replaced by selective media containing 6 μg/ml of puromycin for stable selection of transfected cells. Single colonies resistant to puromycin were isolated. Western-blots were run with cell lysates from these clones to confirm silencing of the cdc37 gene using a 1:1000 dilution of an anti-cdc37 (V367) antibody (Cell Signaling Technology Inc, MA, USA). BACE-1 expression and APP-CTF levels were monitored in cdc37 knock-down HEK293 APPsw cells and HEK293 APPsw cells transfected with a scrambled control shRNA vector as described above.
In order to inhibit HSP90, HEK293 APPsw overexpressing cells were treated for 24 hours with 10 and 20 μM of gedunin (Tocris, MI, USA), a known HSP90 inhibitor which has been shown to inhibit HSP90 with an IC50 ranging from 3 μM to 8 μM in function of the cell type used . Following 24 hours treatment with gedunin, cellular lysates were prepared and assayed for BACE-1 expression and APP-CTF levels by western blotting as described above.
The effect of celastrol on the disruption of the cdc37-HSP90 complex was tested in HEK293 cells overexpressing APPsw. Briefly, HEK293 APPsw cells were treated with PMA 100 ng/ml and celastrol 5 μM for 3 hours. Cellular lysates were prepared as described above and immunoprecipited overnight at 4°C with a 1:100 dilution of an HSP90 antibody (Cell Signaling Technology Inc, MA, USA). Protein A sepharose (GE Healthcare, NJ, USA) was used to pull down the immunoprecipitated complex which was denatured and analyzed by westernblotting for the presence of cdc37.