Ca 074 methyl ester

Human neuroblastoma SK-N-SH cells overexpressing wild-type APP695 (SK-N-SH APPwt) were a gift from Dr. Dennis Selkoe (Boston, MA, USA). SK-N-SH APPwt cells were grown in DMEM, plus10% fetal bovine serum (Hyclone, Los Angeles, CA, USA), 100 U/ml penicillin/streptomycin. Also, cells were supplemented with 200 μg/ml G418. Cell cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2 and passed every 2–4 days based on 85% confluence. P.g-LPS was obtained from Invivo Gen (San Diego, CA, USA), and applied to the supernatant of SK-N-SH APPwt cells with the final concentration 1 μg/ml for a total of 7 days to mimic CP in vitro (P.g-LPS was incubated for 4 days initially. At confluence, SK-N-SH APPwt cells were passaged and new P.g-LPS were applied to the supernatant immediately and incubated for another 3 days). For Cathepsin B inhibition, a final concentration of 75 μM CA-074 methyl ester (Sigma-Aldrich, USA) was applied to the supernatant of SK-N-SH APPwt cells 1 h before P.g-LPS. For Cathepsin B activation, IL-6 (10 ng/ml, Genscript, Z03134), IL-1β(100 pg/ml, Genscript, Z02978), TNF-α (10 ng/ml, Genscript, Z01001) and recombinant Human CRP protein (1 mg/L, Abcam ab171471) were applied to SK-N-SH APPwt cells for 24 h. For TNF-α inhibition, pomalidomide (2 μM, Selleck, S1567) was applied to P.g-LPS treated SK-N-SH APPwt cells 24 h before cell harvest.

ALL cell lines were obtained from DSMZ (Braunschweig, Germany). Cells were cultured in RPMI 1640 medium (Life Technologies/Thermo Fisher Scientific, Darmstadt, Germany). Media were supplemented with 10% FCS (fetal calf serum) (Life Technologies/Thermo Fisher Scientific), 1 mM pyruvate (Life Technologies/Thermo Fisher Scientific), 25 mM HEPES (Life Technologies/Thermo Fisher Scientific) and 1% penicillin/streptomycin (Life Technologies/Thermo Fisher Scientific). The Smac mimetic BV6 was kindly provided by Genentech, Inc. (South San Francisco, CA, USA). The glucocorticoid Dexa was purchased from Sigma-Aldrich (Steinheim, Germany), the caspase inhibitor zVAD.fmk from Bachem (Heidelberg, Germany), the proteasome inhibitor Bortezomib from Selleckchem (Houston, TX, USA) and cycloheximide (CHX), E64d, CA-074 methyl ester, chloroquine, pepstatin A and Bafilomycin A from Sigma-Aldrich. All other chemicals were purchased from Sigma-Aldrich (Steinheim, Germany) or Carl Roth (Karlsruhe, Germany) unless indicated otherwise.

Ricin B-chain was obtained from Vector Labs (Burlingame, CA), HEPES, α-lactose monohydrate, trypsin, pronase, bafilomycin A1, pepstatin A and CA074 methyl ester came from Sigma-Aldrich (St. Louis, MO). [³H]Leucine was purchased from GE Healthcare (Princeton, NJ), Na₂³⁵SO₄ and L-[³⁵S]Methionine came from Hartmann Analytic (Braunschweig, Germany). The mouse monoclonal anti-HA antibodies were obtained from Covance Research Products (Denver, CO), rabbit anti-BACE from Merck (Whitehouse Station, NJ), rabbit anti-Ricinus Communis-Lectin, and mouse anti-α-tubulin came from Sigma-Aldrich, whereas mouse monoclonal anti-ricin A-chain were purchased from Serotec (Oxford, UK). The mouse anti-calnexin were obtained from BD Biosciences (Palo Alto, CA), anti-calreticulin from BioSite (San Diego, CA). The rabbit anti-His antibodies came from Santa Cruz Biotechnology (Santa Cruz, CA). The secondary anti-rabbit HRP and anti-mouse HRP antibodies, were obtained from Sigma-Aldrich, whereas anti-rabbit Alexa555 and anti-mouse Cy-3 were obtained from Jackson laboratories (Bar Harbour, MA).