The triple-negative breast cancer cell line MDA- MB-231 was purchased from ATCC (Manassas, VA, USA) and routinely grown as monolayers at 37°C, in a humidified atmosphere with 5% CO₂, in minimum essential medium (MEM) (Sigma-Aldrich, Saint-Quentin Fallavier, France) supplemented with 10% fetal calf serum (FCS) (Gibco; Invitrogen, Cergy Pontoise, France), 20 mM Hepes, 2 mM L-glutamine, 100 U/ml penicillin/streptomycin (Sigma-Aldrich).

MDA-MB-231 cells were grown in T75 cm² flasks until reaching subconfluency. A set of 7×10⁸ cells were treated, or not, with 100 μM DHA (Sigma-Aldrich Chimie S.a.r.l.) for 24 h. After treatment, cells were rinsed three times with 10 ml MEM and twice with 20 mM phosphate buffer pH 7.4 containing 150 mM NaCl. Cells were then detached with 2 ml of Versene (Sigma-Aldrich) and centrifuged at 200 × g for 10 min at 4°C. The supernatant was discarded and the cell pellet stored at −70°C. The membrane preparations were carried out on ice and at 4°C according to Venkateswaran et al (25). Briefly, the cell pellets were defrosted and homogenized in 3 ml buffer A composed with 20 mM Hepes, 200 mM sucrose and 5 mM EDTA. The suspension was then transferred in a 7 ml Dounce and cells were disrupted with 40 strikes of pestle. The homogenate was centrifuged at 1,000 × g for 20 min at 4°C in order to pellet the nuclei. The supernatant containing the membranes was transferred in a tube for centrifugation. Prior to this step, four volumes of buffer B composed of 20 mM Hepes, 1 mM CaCl₂, 1 mM MgCl₂ (all from Sigma-Aldrich). and 100 mM NaCl were added to the supernatant to decrease buffer density and to allow the best ionic environment for membrane proteins. The mixture was centrifuged at 60,000 × g for 90 min at 4°C in order to obtain crude membrane pellets. The supernatants were removed and the pellets suspended in 500 μl buffer B and washed twice in the same conditions. Aliquots of the pellets were taken for a protein assay using Bradford’s method (Bio-Rad, Marnes-la-Coquette, France) with BSA (Sigma-Aldrich) as standard. Aliquots of 100 μg membrane proteins were stored in Eppendorf tubes and centrifuged at 20,000 × g for 60 min at 4°C. The pellets were then solubilized with 50 μl of a lysis buffer suitable for isoelectric focusing (IEF) (urea 7 M, thiourea 2 M, CHAPS 2%, DTT 40 mM and 0.4% ampholytes 3–10) (GE Healthcare Europe GmbH, Vélizy-Villacoublay, France) containing 1% ASB-14 (Sigma-Aldrich), and stored at −70°C.

A total of 100 μg of membrane proteins prepared as described above were lysed with 50 μl of lysis buffer as described above and processed for IEF by incubation for 1 h in 2 μl of 200 mM tributylphosphine (TBP) (Sigma-Aldrich) followed by incubation for 1.5 h with 5 μl 200 mM iodoacetamide (IAM) (GE Healthcare). At this stage, samples were loaded for IEF. Three independent experiments were carried out in duplicate.

IEF was performed using Ettan IPGphor 3 apparatus using 7 cm strips with pH 3.0–10.0 (both from GE Healthcare Europe GmbH). Strips were rehydrated overnight at room temperature according to the manufacturer’s instructions with DeStreak Rehydration Solution containing 0.4% ampholytes pH 3.0–10.0 (GE Healthcare). The samples (100 μg) were cup-loaded near the anode of the IPG strips and three drops of mineral oil were introduced in the cups. Then, the tray was filled with mineral oil. The run was defined as follows: step at 500 V for 500 Vh, gradient to reach 3,000 V for 5,000 Vh, step at 3,000 V for 12,000 Vh, step 1,000 V for 1,000 Vh. Once the IEF was completed, the strips were processed for SDS-PAGE after equilibration in urea 6 M, PlusOne Glycerol 30% w/v, SDS 2% w/v (Bio-Rad), 0.125 M Tris, 0.1 M HCl containing 50 mM DTT (first equilibration step; Sigma-Aldrich) and 150 mM IAM (second equilibration step; GE Healthcare), and consisting in two baths of 20 min each. The strips were placed at the top of 12% acrylamide-bisacrylamide gels and maintained in position with 2 ml of stacking gel. The run was performed with a PROTEAN 3 apparatus (Bio-Rad) at a constant power of 8 W until the Bromophenol Blue (Merck S.A., Lyon, France) reached the bottom of the gels. Gels were washed twice for 5 min and stained with Imperial Blue^® (Fisher Scientific, Illkirch-Graffenstaden, France) according to manufacturer’s instructions. Three independent experiments were performed in duplicate.

The 2-D gels were scanned with a GS-800 densitometer (Bio-Rad). Spot detection, quantification and analysis were performed with the SameSpots^® v4.1 analysis software (Nonlinear Dynamics, Ltd., Newcastle upon Tyne, UK). Following linearization towards a reference gel chosen among the experimental gels, they were grouped either as control or treated. Each group was the result of three independent experiments performed in duplicate. Spot detection and quantification were determined and a difference was considered to be significant, due to the staining method used, when a 1.5-fold increase or decrease at least was reached. Statistics using ANOVA were given with the in-built statistical software.

The protein spots differentially expressed were excised manually and washed five times for 6 min with 100 μl water. Then the gel spots were soaked in acetonitrile and dried under vacuum. The gel pieces were rehydrated in a reduction buffer [ammonium bicarbonate 100 mM (Sigma-Aldrich), DTT 10 mM (GE Healthcare)] for 1 h at 56°C and 5 min at room temperature. After removing this buffer, they were incubated with an alkylation buffer (ammonium bicarbonate 100 mM, IAM 55 mM) for 45 min at room temperature and protected from light. Then, they were washed in a 25-mM ammonium bicarbonate buffer followed by acetonitrile (Merck S.A.) and finally dried under vacuum. The gel pieces were rehydrated in 100 μl of 25 mM ammonium bicarbonate and incubated with 125 ng of Trypsin Gold (Mass Spectrometry Grade; Promega France, Charbonnières-les-Bains, France) for 1 h on ice. The trypsin digestion was performed for 12 h at 37°C after addition of 30 μl of 25 mM ammonium bicarbonate.

Mass spectrometry analyses were performed using an Ultra flex™ II MALDI-TOF/TOF instrument (Bruker Daltonics, Bremen, Germany). MALDI target plate (AnchorChip™; Bruker Daltonics) was covered with extracted peptides mixed-up with α-cyano-4-hydroxycinnamic acid matrix (0.3 mg/ml in acetone:ethanol, 3:6 v/v). The molecular mass measurements were obtained as previously described (26). Database searches, through Mascot v.2.2.1 (Matrix Science, Ltd., London, UK), using combined PMF and PFF datasets were performed against the UnitProt 2013-06 database (2013-06-17) via ProteinScape 2.1 (Bruker Daltonics). A mass tolerance of 75 ppm and one missing cleavage site for PMF and MS/MS tolerance of 0.5 Da and one missing cleavage site for MS/MS search were allowed. Carbamidomethylation of cysteine and oxidation of methionine residues were also considered. Relevance of protein identities was judged according to the probability-based MOWSE score calculated with a P-value of 0.05 (P≤0.05).

A siRNA (Eurogentec S.A., Seraing, Belgium) directed against KRT1 was used and defined by (GGA-UGU-GGA-UGG-UGC-UUA-U55) for the forward strand and (AUA-AGC-ACC-AUC-CAC- AUC-C55) for the reverse. In addition a control siRNA (NEG) provided by the manufacturer was used. The different siRNA were rehydrated with ultrapure water to obtain a concentration of 20 μM. In a 24-well plate, 4×10⁵ living cells/well were seeded. After 12 h, the medium was removed and rinsed twice with 1 ml/well of Opti-MEM. Then 950 μl of Opti-MEM were added with 50 μl of a mixture containing Lipofectamine (Invitrogen) with or without the appropriate siRNA. To form the mixture, 2.5 μl siRNA at 20 μM were mixed with 22.5 μl Opti-MEM, and apart, 8.32 μl Lipofectamine were homogenized with 16.68 μl Opti-MEM. The two solutions were then mixed. After 10 min of incubation at room temperature the mixture was transferred to culture wells. Incubation was for 4 h at 37°C with 5% CO₂. The medium was discarded and replaced with 1 ml Opti-MEM containing 5% FCS with or without 100 μM DHA. After 24 h, cells were harvested for invasion assay.

Invasion assays were done in 12-well Boyden microchambers (Transwell^®; Fisher Scientific) with 8-μm pore membranes. Matrigel^® (100 μl; BD Biosciences, Le Pont de Claix, France) at 10% in MEM were introduced in the upper chamber and dried overnight at 37°C. Cells treated or not for 24 h as described in the previous section were dissociated with Versene and counted by using a Malassez hemocytometer. Living cells (2×10⁵) treated or not in 400 μl MEM supplemented with 0.5% FCS and 1% BSA were then loaded into the upper chamber. A volume of 800 μl of MEM with 0.5% FCS, and 1% BSA was introduced into the lower chamber. After incubating for 24 h, the Transwell^® was rinsed with PBS, and the Matrigel^® was scraped off the upper surfaces of the membranes. The cells remaining on the underside of the membrane were fixed for 30 min at −20°C in methanol, then stained with Hoechst stain (H6024; Sigma-Aldrich), and mounted on glass slides with glycerol for fluorescence microscopy (Merck S.A.) before counting (15 fields/membrane) under a UV microscope (Biomed with fluorescence equipment; Leica, Rueil-Malmaison, France). Light and fluorescent micrographs were taken with the Lasez software (Leica). Three independent experiments were performed in duplicate.

Experiments were performed with 5×10⁴ cells/chamber on a 16-chamber slide (Fisher Scientific) overnight. Then 200 μl of the Lipofectamine mixture with or without siRNA were added for 4 h after medium withdrawal and rinsing with 500 μl Opti-MEM. Then 200 μl Opti-MEM containing 5% SVF with or without 100 μM DHA were added for 24 h. Chambers were rinsed with PBS and cells fixed with ethanol-methanol-ultrapure water (1:1:2) for 1 h at −20°C. Cells were treated for endogenous peroxidase with 100 μl PBS containing 3% H₂O₂ 20 vol, for 10 min at room temperature. Then the medium was discarded and 200 μl of PBS containing 5% BSA was added for 1 h followed by an incubation of 2 h with 100 μl mouse monoclonal anti-KRT1 antibody (Mab 191–05; Diagnostic BioSystems, Inc., Hague, The Netherlands) at 1/500 diluted in PBS with 0.5% BSA. Cells were rinsed three times with 200 μl PBS 0.5% BSA. HRP anti-mouse antibody (Sigma-Aldrich) at 1/200 in PBS 0.5% BSA was added for 1 h. Cells were rinsed three times with PBS 0.5% BSA and three times with PBS prior HRP revelation by the adjunction of 100 μl diaminobenzidine (DAB) prepared in 10 ml water with 20 μl H₂O₂ 20 vol and counterstained with Hoechst staining. Slides were washed with water and mounted with glycerol for fluorescence microscopy. For each chamber, 10 randomized fields were photographed and analyzed with the Quantity One software (Bio-Rad). Staining intensity in one field was divided by the number of nuclei observed by Hoechst staining in the same field in order to have the average value of KRT1 immunoreactivity. Three independent experiments were performed in duplicate.

Statistical analyses for cell culture were performed using KyPlot^® (KyensLab, Inc., Tokyo, Japan) for a one- way ANOVA followed by a Dunnett’s test to compare untreated or control cells with the treated one. P-value of <0.01 and <0.001 respectively, indicates statistically significant result. In the figures shown as ^**P<0.01 and ^***P<0.001 Statistics for 2-DE are described in the corresponding paragraph.

MDA-MB-231 cells treated or not with 100 μM DHA for 24 h were processed to obtain membrane extracts. After 2-DE of membrane proteins (from both DHA-treated and control cells), it appears that only a few membrane proteins displayed at least a 1.5-fold differential expression pattern (Fig. 1). Four proteins were found upregulated by DHA treatment compared to the control, and one protein was down-regulated (Table I). Mass spectrometry for spot no. 19 was not possible due to insufficient protein quantity and for spot no. 63 it was a mixture of proteins. Spots nos. 70, 110 and 219 were identified (Fig. 2; Table II) by microsequencing using coupled mass spectrometry as being KRT1 (UniProt ID P04264), catalase (UniProt ID P04040) and lamin-A/C (UniProt ID P02545), respectively.

The increase of KRT1 in DHA-treated cells was confirmed by immunocytochemistry (Fig. 3). KRT1 was present in the cytoplasm of untreated (Fig. 3A) and treated (Fig. 3B–D) cells. KRT1 labeling was quantified by using Quantity One software (Bio-Rad). The result was then subtracted by the blank of a similar surface without cells and divided by the number of nucleus present in the field. Thus, the average KRT1 quantification corresponded to a single cell expressed as a percentage (Fig. 3E). The result indicated that control siRNA (NEG) had no effect on the level of KRT1 in the DHA-treated cells and in the control. In the cells treated with DHA and siRNA against KRT1, we observed a level of KRT1 that was reduced nearly to what was observed in the control (Fig. 3E).

Using Matrigel^® in Boyden chambers, DHA was shown to reduce the invasive potential of MDA-MB-231 cells (Fig. 4). In order to investigate the role of the differentially expressed KRT1 protein, siRNAs were used in the invasion assay in presence or absence of DHA. siRNA against KRT1 was able to restore a percentage of invasive cells similar to the control level while the control siRNA NEG was inefficient (Fig. 4). This indicated the involvement of KRT1 in the DHA-induced decrease of MBA-MB-231 cell invasiveness.