Kifunensine

Arabidopsis plants were grown under long‐day conditions (16 h light/8 h dark) at 22°C. T‐DNA insertion lines os9, sel1l and the mns45 mutants were described recently (Hüttner et al., 2012, 2014b). Transgenic Arabidopsis plants were generated by floral dipping and subsequent selection of seedlings on hygromycin‐containing 0.5 ×  Murashige and Skoog (MS) medium. For treatment with kifunensine, cut leaves from 4–5‐week‐old soil grown plants were incubated for 24 h in 0.5 ×  MS medium supplemented with 1% sucrose and 50 μM kifunensine (Santa Cruz Biotechnology). For all transformed constructs at least 12 independent Arabidopsis plants were initially analyzed. In the subsequent generations, kifunensine treatment was repeated for selected independent lines. Treatment with other inhibitors was done with 12‐day‐old Arabidopsis seedlings in the same way by adding 100 μg/ml cycloheximide (Sigma‐Aldrich, Vienna, Austria). N. benthamiana plants were grown on soil under long‐day conditions at 25°C. For inhibitor treatments in N. benthamiana, 20 μM kifunensine were infiltrated into leaves together with agrobacteria carrying plasmids for transient expression of proteins.

Arabidopsis thaliana plants were grown under long-day conditions (16-h light/8-h dark) at 22°C. The os9, sel1l, mns45, and gpi8-1 mutants (R42Q exchange in GPI8) were described previously (Hüttner et al., 2012 (link), 2014a (link); Bundy et al., 2016 (link)). Transgenic plants were generated by floral dipping and subsequent selection of seedlings on hygromycin-containing 0.5× Murashige and Skoog medium. For treatment with kifunensine, 7- to 12-d-old seedlings were incubated for 24 h in 0.5× MS medium supplemented with 1% (w/v) sucrose and 50-µM kifunensine (Santa Cruz Biotechnology). For the induction of ER stress, seedlings were incubated 18 h in 5 mM AZC (Sigma-Aldrich) or 2 mM DTT (Sigma-Aldrich). CHX treatment was done with 12-d-old Arabidopsis seedlings by adding 100 µg/mL CHX (Sigma-Aldrich). Nicotiana benthamiana plants were grown on soil under long-day conditions at 25°C. For inhibitor treatments in N. benthamiana, 50-µM kifunensine was infiltrated into leaves together with Agrobacterium tumefaciens carrying plasmids for transient expression of proteins.

Unless otherwise specified, cells were grown and cultured at a concentration of 1 × 10⁶ cells/mL of standard culture media (RPMI 1640 + 10% FCS, 1% penicillin/streptomycin, 1% HEPES, 1% non-essential amino acids) at 37 °C in 5% ambient CO₂. For glycosidase treatments, cultures were supplemented with kifunensine (Santa Cruz sc-201634) at a concentration of 16 ng/mL. Jurkat, Nalm6, and OCI-Ly10 parental cell lines were obtained from ATCC. Primary human T cells were purified from human peripheral blood mononuclear cells, obtained commercially from Miltenyi (Catalog #150-000-452).