Cycloheximide (chx)

Total RNA was isolated from S2 cells and unfractionated embryos using TRIzol^® (Invitrogen) and miRVana miRNA isolation kit (Ambion, Waltham, MA, USA), respectively. Ribonucleoprotein complexes were fractionated according to a previously published procedure with minor modifications [22 (link)]. Briefly, embryos (0.2 g) or S2 cells (3 × 10⁷) were homogenized in 5 mL lysis buffer ((100 mM NaCl, 10 mM MgCl₂, 30 mM Tris-HCl (pH 7), 1% Triton X-100, 1% NaDOC, 100 µg/mL cycloheximide (Applichem, Darmstadt, Germany) and 30 U/mL SUPERase.In^™ RNase Inhibitor (Ambion, Waltham, MA, USA)) and incubated on ice for 8 min. The homogenates were centrifuged at 12,000× g at 4 °C for 8 min. Two-mL supernatant was loaded onto 5–70% (w/v) sucrose gradients (100 mM NaCl, 10 mM MgCl₂, 30 mM Tris-HCl (pH 7), 200 U SUPERase. In^™ RNase inhibitor (Ambion, Waltham, MA, USA)) and centrifuged at 27,000 rpm for 2 h 55 min at 4 °C in a Beckman SW28 rotor. Fractions were collected using an ISCO density gradient system. Fractions were then pooled into 4 sub-groups based on their A₂₅₄ readings; mRNP, 60S, monosome and polysome. Total RNA was extracted from the fractions as previously described [22 (link)]. RNA quality was assessed by 2100 bioanalyzer using RNA 6000 Nano Kit based on manufacturer’s instructions (Agilent, Santa Clara, CA, USA).

Polysome profiles were obtained according to a previously published procedure (Göktaş et al., 2017 (link)). Briefly, cell lysis (3×10⁷ cells) was carried out in 5 mL lysis buffer [(100 mM NaCl, 10 mM MgCl₂, 30 mM Tris-HCl (pH 7), 1% Triton X-100, 1% NaDOC, 100 μg/mL cycloheximide (Applichem) and 30 U/mL SUPERase.IN RNase inhibitor (Ambion)], and the lysate was incubated on ice for 8 min. The cell debris and nuclei were removed by centrifuging the homogenates at 12,000 g at 4 °C for 8 min. Two-mL supernatant was loaded onto 5%–70% (w/v) sucrose gradients [100 mM NaCl, 10 mM MgCl₂, 30 mM Tris-HCl (pH 7), 200 U SUPERase.IN RNase inhibitor (Ambion)] and centrifuged at 27,000 rpm for 2 h 55 min at 4 °C in a Beckman SW28 rotor. Fractions were collected using Teledyne ISCO’s density gradient fractionation system (NE, USA) while recording the absorbance at A₂₅₄ to obtain the polysome profiles.

Classical microbiological analysis was performed in olive samples to enumerate the main microbial groups implicated in table olive fermentations [23 (link)], i.e., TVC, LAB, yeasts and molds, and Enterobacteriaceae. For this purpose, olives were removed from the brine and 25 g of olive flesh was aseptically cut and homogenized in 225 mL sterile ¼ Ringer’s solution (Stomacher 400 circulator, Seward Limited, Norfolk, United Kingdom) for 60 s at room temperature. The appropriate decimal dilutions were poured or spread on the following growth media: (i) Tryptic Soya Agar (TSA, 4021502, Biolife, Milan, Italy ) for TVC enumeration, incubated at 25 °C for 48–72 h; (ii) de Man-Rogosa-Sharpe agar (MRS LAB233, LABM) for the enumeration of LAB, supplemented with 0.05% (w/v) cycloheximide (AppliChem, Darmstadt, Germany), overlaid with the same medium and incubated at 30 °C for 48–72 h; (iii) Rose Bengal Chloramphenicol Agar (RBC Agar, BK151HA, Biokar diagnostics, Allone, France) for the enumeration of yeasts and molds, incubated at 25 °C for 48 h; and (iii) Violet Red Bile Glucose Agar (VRBGA, CM0485, Oxoid, Hampshire, United Kingdom) for the enumeration of Enterobacteriaceae, overlaid with the same medium and incubated at 37 °C for 24 h. The results were log transformed and expressed as log CFU/g.

Polysome profiling was performed according to a published procedure [23 (link)]. Following centrifugation at 1200 RPM for 10 min, the cells were homogenized in lysis buffer [100 mM NaCl, 10 mM MgCl₂, 30 mM Tris-HCl (pH 7), 1% Triton X-100, 1% NaDOC, 100 µg/mL cycloheximide (Applichem, Darmstadt, Germany) and 30 U/mL RNase Inhibitor (Promega, Madison, WI, USA)]. The cell pellets were sheared to homogenization by passing the lysate through a 26 G needle at least 15 times and incubated on ice for 8 min. The lysates were then centrifuged at 12,000× g at 4 °C for 8 min. The supernatants were layered over 5–70% (w/v) sucrose gradients [100 mM NaCl, 10 mM MgCl₂, 30 mM Tris-HCl (pH 7.0), 200 U RNase Inhibitor (Promega, Madison, WI, USA)] prepared by using an ISCO Teledyne (Lincoln, NE, USA) density gradient fractionation system and centrifuged at 27,000 RPM for 2 h 55 min at 4 °C in a Beckman SW28 rotor (Beckman Coulter, Brea, CA, USA). Fractions were collected using the ISCO Teledyne density gradient fractionation system while reading absorbance at A₂₅₄. Fractions were pooled as mRNP, monosomal, light and heavy polysomal sub-groups based on A₂₅₄ readings. The total RNA was phenol-extracted from fractions by using phenol-chloroform-isoamyl alcohol (25:24:1) (Applichem, Darmstadt, Germany).