*2.4. Protein Extraction and Quantification*

Proteins of wt and of the two mutant lines, untreated and treated (80 mg L−<sup>1</sup> CdS QDs), were extracted. Frozen samples were ground to powder in liquid nitrogen using a mortar and pestle; 1 g aliquot was suspended in 6 mL of extraction buffer: 700 mM sucrose, 500 mM Tris-HCl, pH 7.5, 50 mM ethylenediaminetetraacetic acid (EDTA), 100 mM KCl, 2% dithiothreitol (DTT), 0.1% Protease Inhibitor Cocktail (Sigma-Aldrich), vortexed and mixed for 10 min on ice. An equal volume of 500 mM Tris-HCl buffered phenol was added, and the solution was mixed at room temperature for 10 min [30,31]. The samples were

centrifuged for 10 min at 5500× *g* and at 4 ◦C. The phenolic phase was collected in a new tube and back-extracted with 3 mL of extraction buffer. Proteins were precipitated from the phenolic phase overnight at −20 ◦C by adding five volumes of 0.1 M ammonium acetate (J.T. Baker, Deventer, The Netherlands) saturated in methanol. Precipitated proteins were centrifuged for 30 min at 5500× *g* and at 4 ◦C, the pellet was washed with cooled methanol and then with cooled acetone. After each washing step, the sample was centrifuged for 5 min at 5500× *g* and at 4 ◦C. Finally, the pellet was dried using a Speed Vac Concentrator 5301 (Eppendorf AG, Barkhausenweg, Hamburg, Germany).

The pellet was dissolved in 300 μL of isoelectofocusing (IEF) buffer containing 9 M urea, 4% 3-[(3-cholamidopropyl) dimethylamino]-1-propanesulfonate (CHAPS), 50 mM DTT, 0.001% protease inhibitor cocktail, 1% carrier ampholyte mixtures (pH 3–10, BioRad, CA, USA). Protein quantification was evaluated according to a modified Bradford assay [32] based on the acidification of the sample buffer with 20 mM HCl. Bovine serum albumin (BSA) was used as standard.

## *2.5. 2D Gel Electrophoresis*

The proteins mixtures were resolved by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). For proteins separation in the first dimension (IEF), 400 μg of proteins of each sample were loaded onto 11-cm ReadyStrip pH 5-8 IPG strips (BioRad, CA, USA) which had been rehydrated overnight with 250 μL IEF buffer containing the sample. Proteins were focused by PROTEAN® i12™ IEF System (BioRad, CA, USA) applying 250 V (60 min), 1000 V (60 min), 8000 V (2 h) and 8000 V up to 35,000 V/h.

After IEF, the strips were incubated 15 min in 3 mL of reducing buffer containing 6 M urea, 2% *w*/*v* DTT, 0.375 M Tris-HCl (pH 8.8), 20% *w*/*v* glycerol, 2% *w*/*v* SDS and for 15 min in 3 mL of alkylating buffer containing 6 M urea, 2.5% w/v iodoacetamide, 0.375 M Tris-HCl (pH 8.8), 2% *w*/*v* glycerol. The second dimension (SDS-PAGE) was performed using a CriterionTM Dodeca™ cell (BioRad, CA, USA) and 12% Criterion™ XT Bis-Tris gels (BioRad, USA) in 1 M MOPS (3-(N-morpholino)-propanesulfonic acid) buffer (1 M Tris, 20 mM EDTA and 2% *w*/*v* SDS).

2D gels were stained with QC Colloidal Coomassie G-250 (BioRad, CA, USA) and were scanned with a ChemiDocTM Imaging System (BioRad, CA, USA). Gel analysis was performed using PDQuest software (BioRad, CA, USA). Spot detection and matching between gels were performed automatically, followed by manual verification. The spot densities were normalized by local regression method and followed by a calculation against the whole gel densities. The percentage density of every spot was averaged over three replicate gels, and Student's *t*-test analysis (*p* < 0.05) was carried out to find statistically significant differences in protein abundances. Statistically significant spots were then excised from the gels using an EXQuest Spot Cutter (BioRad, CA, USA), destained by soaking the pieces of acrylamide in a 1:1 solution of 100 mM ammonium bicarbonate/acetonitrile for 30 min, and the proteins were hydrolyzed with trypsin at 37 ◦C overnight [33].
