# Modulation of Ricin Intoxication by the Autophagy Inhibitor EACC
## 2 Results
### The TBK1 Inhibitor MRT68601 Increases Ricin Transport to the Golgi and Sensitizes Cells to Ricin  
As described in the introduction, autophagy initiation is dependent on the kinase TBK1 [8], and we therefore tested whether an inhibitor of this kinase would affect the protective effect seen with EACC on HEp-2 cells. As shown in Figure 5, MRT68601 (25 \(\upmu\)M) surprisingly induced a strong sensitization to ricin. However, EACC was still able to induce  
Figure 4: Release of ricin A-chain after EACC treatment. HEp-2 cells were incubated for 1 h with \({}^{35}\)SO\({}_{4}\)\({}^{2-}\) and then EACC was added to some of the cells (5 \(\upmu\)M), and the incubation was continued for 2 h more before further addition of RS1 and MG132 (10 \(\upmu\)M) to all the cells. After 3 h of additional incubation, N-ethylmaleimide (1 mM) was added to prevent further reduction of RS1, and surface-bound toxin was removed with lactose. Then the cells were lysed, and an anti-ricin A-chain antibody was used for immunoprecipitation. The precipitates were then washed twice with PBS containing TritonX-100 (0.35%), resuspended in Laemml sample buffer with and without 100 mM DTT, and boiled for 5 min before being run on SDS-PAGE. Further processing and quantification were performed as described in Materials and Methods. In (**A**) is shown one representative example of such an experiment where the samples to the left were run on a non-reducing gel (\(-\)DTT) and the samples to the right on a reducing gel (\(+\)DTT). (**B**) shows quantification of the release of A-chain from independent experiments (\(n=3\)); **\(p\leq 0.005\)**. a good protection in the presence of MRT68601. A representative experiment is shown in Figure 5A, and the graphs in Figure 5B,C show a quantification of the protection and sensitization from independent experiments. As also demonstrated (Figure 5D), none of the treatments had any major effect on protein synthesis. As expected, the ability of MRT68601 to sensitize cells to ricin was concentration dependent, as shown in Supplementary Figure S4. To investigate which step of ricin transport was affected by MRT68601, we again used the ricin construct RS2 with both a sulfation site and a glycosylation site, and we investigated what happens both with MRT68601 alone and in combination with EACC. As shown in Figure 6, MRT68601 strongly increased the sulfation of this construct, indicating an increased transport of the toxin to the Golgi apparatus. There was an equally strong increase also in the glycosylation of ricin as indicated by the heavier sulfated band, and the data thus suggested that the efficiency of trans-Golgi to ER transport was unchanged; the increase in transport to the Golgi seemed to be reflected by a similar increase in transport to the ER. Importantly, even in the presence of both MRT68601 and EACC, glycosylation occurred to the same extent as with MRT alone (Figure 6), indicating that even though more ricin entered the ER than in control cells, EACC was able to inhibit transfer of ricin into the cytosol. Additionally, in HeLa cells, sulfation experiments showed that MRT68601 in a concentration-dependent manner increased ricin transport to the Golgi apparatus (Figure S5). Figure 5: MRT68601 strongly sensitized cells to ricin, an effect counteracted by EACC. HEp-2 cells were treated with 25 \(\upmu\)M MRT68601 or DMSO (0.1%) for 30 min prior to addition of 5 \(\upmu\)M EACC or DMSO. Following additional 2 h incubation with the inhibitors, the cells were incubated with increasing concentrations of ricin for 3 h, before protein synthesis was measured. One representative experiment is shown in (**A**). (**B**) Fold protection was calculated as an increase in IC50 in EACC-treated samples compared to control (DMSO-treated cells). (**C**) Fold sensitization was calculated for the samples treated with MRT alone. (**D**) Protein synthesis in the cells treated with the inhibitors for 5.5 h (2.5 h + 3 h when the toxin was added to other wells). \(n\) = 4.