Bortezomib bz

BC3, BCBL1, Raji and B95-8 cell lines were treated with bortezomib (BZ) (Santa Cruz Biotechnology Inc.) at 20 nM for 0, 4, 6, 24 hrs or mock treated. A dose-response experiment with 0-10-20 nM (for 24 hours) was also performed in the same cell lines. In some experiments, BC3 were treated with Thapsigargin (5 μM) for 24 hrs. To evaluate the role of autophagy in viral replication, BC3 and Raji cell lines were cultured with bortezomib (20 μM) (Santa Cruz Biotechnology Inc.) in the presence or in the absence of chloroquine (10 μM) (Sigma Aldrich) or with 3-Methyladenine (3-MA) (5 mM) (Santa Cruz Biotechnology Inc.) for 24 hrs^{32 (link),33} . To further investigate autophagy, siRNAATG5 experiments were performed on the same cell line, as previously reported^{15 (link)}.
In order to investigate the role of JNK, BC3 and B95-8 cell lines were pre-treated with SP600125 (SP, JNK inhibitor) (Santa Cruz Biotechnology Inc.) at 20 μM or Raji and BCBL1 cells were transfected with HA-JNK-APF nonphosphorylatable mutant of JNK (DN-JNK) plasmid or control vector^{13 (link)}, and then cultured in presence of bortezomib (BZ) (20 nM) for 24 h.
In some experiments, cells were pretreated for 30 min with z-VAD pan caspase inhibitor (50 μM) (Santa Cruz Biotechnology Inc.) before exposure to bortezomib at 20 nM for 24 hours.

Infection experiments of iDCs or mDCs were performed as recently described [82 (link),83 (link)]. Briefly, iDCs or mDCs were harvested and washed in RPMI 1640. Cells (1 × 10⁶–2 × 10⁶) were resuspended in 300–350 µL infection medium (RPMI 1640 supplemented with 20 mM HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; Lonza)) containing a defined amount of HSV-1 or HSV-2 plaque forming units (pfu). For HSV-1 infection, a multiplicity of infection (MOI) of 1 or 2 was used (as indicated), whereas HSV-2 infection experiments were performed using an MOI of 5. As a mock control, cells were treated with infection medium supplemented with the respective amount of MNT buffer (30 mM MES, 100 mM NaCl, and 20 mM Tris). After 1 h of infection at 37 °C and shaking at 300 rpm cells were collected via centrifugation. Subsequently, cells were cultured in DC medium supplemented with 40 U/mL GM-CSF and 250 U/mL IL-4. To block proteasomal degradation either 10 µM MG-132 (Enzo Life Science, Lörrach, Germany) or 2 µM bortezomib (BZ; Santa Cruz Biotechnology, Heidelberg, Germany) were added 1 hpi (hour post infection). For blocking the ubiquitin E1-activating enzyme, 80 µM PYR-41 (Sigma-Aldrich) was added 4 hpi. As solvent control, dimethyl sulfoxide (DMSO; Sigma-Aldrich) was added to the medium.