Silencing of Entamoeba histolytica atg8 gene

E. histolytica strain in which the atg8 gene was silenced (atg8-gs) and the control strain (mock vector transfected) were produced from the G3 strain, as described previously (Picazarri et al., 2015 (link)). Trophozoites of these transformants were maintained axenically in Diamond’s BI-S-33 medium (BIS) (Diamond et al., 1978 (link)) supplemented with 10 μg/mL Geneticin at 35.5˚C. Chinese hamster ovary (CHO) cells were maintained in F12 medium (Sigma-Aldrich, St Louis, MO) supplemented with 10% fetal calf serum (Sigma-Aldrich, St Louis, MO) at 37˚C with 5% CO₂. Paramagnetic Dynabeads, dithiobis succinimidyl propionate (DSP), and OPTI‐MEM I medium were purchased from Thermo Fisher Scientific (Waltham, MA). A complete mini mix was purchased from Roche (Basel, Switzerland). Human serum was purchased from Sigma-Aldrich (St Louis, MO). Anti-HA (11MO), anti-Myc (9E10), and anti-FLAG (clone M2) monoclonal antibodies were purchased from Covance (Princeton, NJ) and Sigma-Aldrich. The production of rabbit polyclonal antibodies against EhAtg8, EhCS1, and EhCP-A5 were previously described (Nozaki et al., 1999 (link); Picazarri et al., 2008 (link); Nakada-Tsukui et al., 2012 (link)). All chemicals of analytical grade were purchased form Sigma-Aldrich unless otherwise stated.