Bortezomib

HeLa, HEK293T, HCT, U2OS, and MCF7 cells were purchased from and authenticated by ATCC. HeLa, HEK293T, U2OS, and MCF7 cells were cultured in DMEM (Biochrom) and HCT cells were cultured in McCoy (Biochrom) supplemented with 10% FCS (Biowest) and 100 µg/ml streptomycin and 100 unit/ml penicillin (Biochrom) at 37 °C on plastic culture flasks (TPP) at saturated humidity and a CO₂ saturation of 5%. Cells were routinely tested for mycoplasma contaminations by PCR.
For DNA damage induction, cells were treated with Doxorubicin (DOX, Sigma-Aldrich) or Etoposide (ETO, Sigma-Aldrich). Staurosporine (STS) and zVAD-fmk (zVAD) was purchased from Enzo Life Sciences.
For lysosomal inhibition, cells were treated with 100 nM Bafilomycin A1 (Biomol), 30 mM NH₄Cl (Merck), or 10 µM Chloroquine (Sigma-Aldrich) for 16 h.
For proteasomal inhibition, cells were treated with 5 µM MG132 (Sigma-Aldrich) or 50 nM Bortezomib (Teva) for 16 h.
Lipofectamine^® LTX with Plus™ (Invitrogen) or Polyethylenimin (PEI) (Polysciences Europe GmbH) was used for plasmid transfection in HeLa and HEK293T cells according to the manufacturer’s instructions. For siRNA transfection, Lipofectamine^® RNAi MAX™ (Invitrogen) was utilized according to the manufacturer’s instructions.

The library consists of 2,491 structurally diverse natural substances, and their derivatives was obtained from InterBioScreen. Sorafenib was obtained from ChemRar. Bortezomib, cisplatin, and 5F-uracil were produced by Teva. Nocodazole and cycloheximide were purchased from Sigma.