# Modulation of Ricin Intoxication by the Autophagy Inhibitor EACC
## 2 Results
### Ricin Is Transported All the Way to the ER in the Presence of EACC  
To investigate which step in ricin entry that was inhibited by EACC, we first measured binding to the cell surface and endocytic uptake by using \({}^{125}\)I-labeled ricin. As shown in Figure 2A, there was no effect on binding to the cell surface, but there was a slight (20%) inhibition of the endocytic uptake. Additionally, there was a small decrease in recycling back to the cell surface and in degradation of ricin (Figure 2B), which could be due to reduced ricin uptake after EACC treatment. Figure 1: EACC protected HEp-2 cells against ricin. HEp-2 cells were treated with EACC for 30 min (**A**) or 2 h (**B**) and subsequently incubated with increasing concentrations of ricin for 3 h, before protein synthesis was measured. Representative experiments are shown (**A**,**B**). (**C**) Fold protection was calculated as an increase in IC50 in EACC-treated samples compared to control (DMSO-treated cells). #: At 10 \(\upmu\)M dose, cells were fully protected against ricin (for up to 1000 ng/mL) and protection could not be calculated. (**D**) Protein synthesis in the cells treated with EACC for 3.5 h (30 min + 3 h when the toxin was added to other wells) or 5 h (2 h + 3 h when toxin was added to other wells). \(n\geq 3\); \({}^{*}p\leq 0.05\). \({}^{**}p\leq 0.005\). Figure 2: The effects of EACC on ricin binding, uptake, and recycling. HEp-2 cells were treated with 0.1% DMSO or 5 uM EACC for 2 h followed by incubation with \({}^{125}\)I-ricin for 20 min. The ricin endocytosis (**A**) and recycling and degradation (**B**) were measured; \(n=3\), as described in Materials and Methods. Endocytosed toxin was measured after 20 min (**A**), and released and degraded toxin was measured after an additional 2 h chase; \({}^{*}p\leq 0.05\). To investigate ricin transport to the Golgi and ER, we used two different ricin constructs, one with a sulfation site in the A-chain (RS1), and another with a sulfation site and a glycosylation site as well (RS2). We added radioactive sulfate to obtain labeling of the toxin constructs (both RS1 and RS2) in the trans-Golgi by sulfotransfareases [12]. There is a shift in the molecular weight of the sulfated A-chain in RS2 when it is modified by ER enzymes. After addition of EACC, sulfation of RS1 was somewhat reduced (Figure 3A), but it was reduced to a similar extent as sulfation of endogenous cellular molecules (total protein sulfation), suggesting that RS1 transport to the Golgi may not be affected much by EACC. It seems unlikely that the very good protection against ricin provided by EACC was due to inhibition of ricin transport to the Golgi. When using the RS2 construct, the challenge was that the radioactive signal from radioactive sulfate became split into two bands (only sulfated and sulfated plus glycosylated) and then became quite weak, especially after addition of EACC (Figure 3B). We therefore added the proteasome inhibitor MG132 to some cells to prevent degradation, and as shown in Figure 3B, the signals then became much stronger. The compound MG132 had in itself no effect on ricin toxicity or on the protection afforded by EACC (Figure S2). Importantly, the data clearly showed that EACC does not prevent the formation of the high molecular weight second band, which was previously demonstrated to be the glycosylated form of the A-chain [12]. Examples of sulfation experiments with RS1 and RS2 and quantification of the bands are shown in Figure 3. The data indicate that ricin is transported all the way to the ER even in the presence of EACC. Thus, an inhibited retrograde transport does not seem to be the explanation for the strong EACC-induced protection. Figure 3: Effect of EACC on sulfation of ricin and endogenous proteins. (**A**) HeLa cells were treated with 0.1% DMSO or 5 uM EACC for 2 h and then were subjected to sulfation assay with ricin-sulf1 (RS1).