*3.9. Formation of F-Actin in AFAT*

We have previously shown that AF leads to the oxidation of cytoskeletal proteins and inhibits the formation of actin filaments (F-actin) [28]. In contrast, cytoskeletal proteins in AFAT are not significantly enriched among OXs according to the PANTHER statistical overrepresentation test (Table S2). In order to confirm this observation, we looked at the level of F-actin in WT trophozoites, acute AF trophozoites, and AFAT. As described previously [28], the F-actin signal in acute AF trophozoites was significantly less intense than that in WT trophozoites. In contrast, the F-actin signal was identical in WT trophozoites and AFAT (Figure 5A,B). These results confirm that the formation of F-actin is impaired in acute AF trophozoites [28], but is not impaired in AFAT.

## *3.10. Overexpression of EhTrxR Does Not Protect E. histolytica Trophozoites against AF*

Overexpression of TrxR in the parasite *Giardia lamblia* has no effect on its resistance to AF [26]. In *E. histolytica*, Debnath et al. found that AF inhibits the amebic TrxR and its reduction, leading to a higher sensitivity of trophozoites to ROS-mediated killing [39]. Our observations regarding the level of TrxR expression, which was the same in WT trophozoites and in AFAT (Table S1), and the fact that Trxs are enriched OXs in AFAT, strongly suggest that *E. histolytica* TrxR is not central to the mechanism of adaptation of the parasite to AF. To test this hypothesis, we overexpressed EhTrxR in *E. histolytica* trophozoites. Overexpression of EhTrxR was confirmed by Western blotting and its level of expression in *E. histolytica* was proportional to the amount of G418 used for selection (Figure 6A–C) [40]. Next, we determined the level of resistance to AF of HA-tagged EhTrxR trophozoites. We observed that the level of resistance to AF of HA-tagged EhTrxR trophozoites did not differ significantly from the level of resistance of the control trophozoites (trophozoites

transfected with pEhExGFP (a kind gift from Dr. Tomoyoshi Nozaki [41])) (Figure 6D). pEhExGFP allows the constitutive expression of the green fluorescent protein (GFP).

**Figure 5.** Formation of F-actin in WT trophozoites, acute AF trophozoites, and AFAT. (**A**) Confocal laser scanning microscopy of F-actin and total actin in WT trophozoites, acute AF trophozoites, and AFAT showed that F-actin was detected using rhodamine-conjugated phalloidin. Total actin was detected using a primary actin antibody and a secondary Cy2-conjugated immunoglobulin G (IgG) antibody. The nuclei (blue) were stained by 4- ,6-diamidino-2-phenylindole (DAPI). (**B**) A computerassisted image was overlaid on the signal emitted by the actin antibody, phalloidin, and DAPI. Fluorescence quantification was performed using Fiji software [38] on 10 trophozoites and the F-actin signal was normalized to the total actin signal. The level of F-actin in WT was arbitrary defined as 1. Data are expressed as the mean ± standard deviation of two independent experiments. The level of F-actin in acute AF trophozoites was significantly different from that in WT and AFAT according to the results of an unpaired Student's *t* test (\*\* *p* value < 0.01). No difference of F-actin level between WT and AFAT was observed according to the results of an unpaired Student's *t* test (*p* value > 0.05).

**Figure 6.** Western blot analysis of *E. histolytica* trophozoites that overexpress a hemagglutinin (HA)-tagged EhTrxR and viability assay. Legend: Protein molecular weight marker (PM). Control trophozoites (lane 1). HA-tagged EhTrxR trophozoites cultivated in the presence of an increasing concentration of G418 (lane 2: 6 μg/mL, lane 3: 30 μg/mL, lane 4: 48 μg/mL). (**A**) Ponceau staining of a nitrocellulose membrane containing cytosolic proteins (40 μg) separated by SDS PAGE of control trophozoites and of HA-tagged EhTrxR trophozoites cultivated in the presence of an increasing concentration of G418. (**B**) Immunodetection of (HA)-tagged EHTrxR with an HA monoclonal antibody (1:500) in HA-tagged EhTrxR trophozoites cultivated in the presence of an increasing concentration of G418. (**C**) Relative quantification of the HA EhTrxR signal following its normalization with the level of total protein in each well with ImageJ software. Normalized values for control trophozoites were taken as 100%. These results are representative of two independent experiments. (**D**) Viability assay. *E. histolytica* trophozoite controls and HA-tagged EhTrxR trophozoites were cultivated in the presence of 2 μM AF for 24 h. The cells were harvested at 400× *g* for 5 min, stained with Propidium iodide, and analyzed by flow cytometry. Flow cytometry was performed using Cyan ADP (Agilent Dako, CA, USA) and data from 10,000 cells were collected for each condition. Data are expressed as the mean ± standard deviation of three independent experiments that were performed in triplicate. The viability of the control trophozoites was defined as 100%. The viability of control trophozoites was not significantly different (ns) from that of the HA-tagged EhTrxR trophozoites according to the results of an unpaired Student's *t* test (*p* value < 0.05). The viability of control trophozoites was significantly different from that of the control trophozoites exposed to AF according to the results of an unpaired Student's *t* test (\*\*\*\* *p* value < 0.001). The viability of HA-tagged EhTrxR trophozoites was significantly different from that of the HA-tagged EhTrxR trophozoites exposed to AF according to the results of an unpaired Student's *t* test (\*\*\*\* *p* value < 0.001).
