*2.7. Classification of OXs According to Their Protein Class and Statistical Overrepresentation Test*

The OXs were classified according to PANTHER Protein Class using the PANTHER Classification System software (http://www.pantherdb.org/, accessed on 28 July 2021) [26]. This classification of proteins derived from PANTHER/X molecular function ontology includes commonly used classes of protein families, many of which are not covered by GO molecular function.

Regarding the statistical overrepresentation test, the online system compares a list of genes of interest (in this work, genes encoding for OXs in *E. histolytica* trophozoites exposed to *L. acidophilus*) to a reference list (*E. histolytica* in database). The *p*-value calculation in the overrepresentation test is calculated automatically based on the number of genes expected in the test list for a particular PANTHER category, based on the reference list.

#### *2.8. Measurement of Cysteine Proteases (CPs) Activity*

CPs activity was monitored by cleavage of the synthetic substrate benzyloxycarbonyll-arginyl-l-arginine-p-nitroanilide (z-Arg-Arg-pNA) (Bachem, Torrance, CA, USA) using a previously described protocol [27] except that DTT was not systematically added to the reaction buffer. Briefly, z-Arg-Arg-pNA was incubated for 0–10 min at 37 ◦C with

*E. histolytica* lysate (40 μg) (prepared in phosphate buffer saline (PBS) nonidet P-40 (1%) (Sigma-Aldrich, Jerusalem, Israel) in 990 μL CP buffer (0.1 M KH2PO4, 2 mM EDTA, pH 7.0). Cleavage of Z-Arg-Arg-pNA substrate were detected at 405 nm in a Novaspec plus spectrophotometer (Sigma-Aldrich, Israel).
