Pes filter

Lactiplantibacillusplantarum cell‐free culture supernatants (CFCS) were prepared from the spent media collected after L. plantarum incubation in cMRS for 24 h at 30°C. CFCS was collected by centrifugation at 4000× g for 10 min at 4°C followed by filtration of the supernatant through a 0.45 μm polyethersulfone (PES) filter (Genesee Scientific, San Diego, CA). To eliminate the effects of differences of pH on yeast inhibition, the CFCS was adjusted with lactic acid (1.3 M) to pH 3.8, the lowest pH reached by L. plantarum after incubation in cMRS (data not shown). S. cerevisiae UCDFST 09‐448 (Golomb et al., 2013 (link)), a strain shown to cause olive tissue damage and spoilage during olive fermentations, was grown in yeast mould (YM) broth (BD, Franklin Lakes, NJ) for 24 h at 30°C with aeration at 250 rpm. Cells were collected by centrifugation at 20 000× g for 5 min at 4°C and then washed twice with PBS. S. cerevisiae UCDFST 09‐448 was then inoculated into 96‐well microtiter plates containing 1:1 ratio of 2X YM and CFCS at a starting OD₆₀₀ of 0.05. OD₆₀₀ was measured in a Synergy 2 microplate reader (Biotek, Winooski, VT) set at 30°C for 24 h aerated every hour by shaking for 10 s before each read. Controls included S. cerevisiae UCDFST 09‐448 incubated in YM and YM supplemented with cMRS (pH 3.8).

Lentiviral particles were produced by co-transfecting the envelope plasmid pCMV-VSV-G (Addgene), packaging plasmid pCMV-dR8.2 dvpr (Addgene), and GIPZ shRNA vectors (GE Dharmacon): E2F1 shRNA (V3LHS_393591), E2F2 shRNA (V3LHS_324068), E2F3 shRNA (V3LHS_325936), HELLS shRNA (V2LHS_155497), or GIPZ lentiviral empty vector shRNA control into HEK293T cells using calcium phosphate transfection method. Supernatants containing lentiviral particles were harvested at 24 and 48 hours post-transfection. Cell debris were cleared by centrifugation at 1600 × g for 10 min at 4° C. Supernatants were then filtered through 0.45μm PES filter (25–223, Genesee Scientific), and concentrated by ultracentrifugation at 23000 RPM for 2 hours at 4° C. Lentiviral particles were resuspended in ice-cold PBS and stored at −80° C. Transduction of target cells were achieved by exposing cells to viral particles in serum-free condition for 6 hours. Puromycin selection was carried out at a concentration of 2 μg/ml.