This protocol is adapted from McClain et al. [18]. After 36 h of siRNA treatment cells were detached from the cell culture plate (polystyrene, cell culture treated) by trypsinization and plated at 150,000 cells/well in a 96-well tissue culture plate; Three hours after cell plating the multiwell plate was centrifuged twice in inverted position (well bottom placed up in a swing out rotor) at 1,000 rpm for 30 min. The cells still adherent to the wells were then processed in two different ways in independent experiments: 1) viable cells were evidenced by a MTT colorimetric method [7] or 2) they were washed twice with PBS, fixed in 3.7% paraformaldehyde in PBS, and stained with Methylene Blue. After the staining the cells were analyzed by digital imaging and cells were counted.

Dlg4/PSD-95 and Epb4.1/3 mRNAs were measured by Real-Time quantitative RT-PCR Sybr Green method, using PE ABI PRISM@ 7,700 Sequence Detection System (Perkin Elmer). The sequences of forward and reverse primers as designed by the Primer Express 1.5 software were: 1) Mouse ROLP 5′-TGAGTACAATGACATCAAGGATGTATCA-3′ and 5′-GATCAAAATCTGGATATAACTCCT TGGT-3′. 2) Dlg4/PSD-95: 5′-CACGAAGCTGGAGCAGGAG-3′ and 5′-GGCCTGAGAGGTCTTC GATG-3′. 3) Epb4.1/3: 5′-TGCAAAGTGACGCTTCTGGAT-3′ and 5′-GTCTACAAGCGTGTG AAACAG-3′. 4) Integrin alpha 1: ITGA1-F AGCAGCAGCAACCGGAAAC and ITGA1-RCA AAGGCGCTCAGGAGGAT. As endogenous control the expression of Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) was examined by quantitative RT-PCR as described above. The mouse GAPDH primers were 5′-TGTGTCCGTCGTGGATCTG-3′ and 5′-GATGCCTGCTTCACCAC CTT-3′. Relative transcript levels were determined by the standard curve method following manufacturer's instructions.

Target sequences for ROLP siRNAs were identified by detecting AA dinucleotides in ROLP mRNA sequence. The downstream 19 nucleotides were compared to the mouse genome database in order to select those with no homology to other genes. siRNAs were synthesized by using the Silencer siRNA Construction Kit (Ambion, UK) according to the manufacturer's instructions. The oligos used were: mROLP Antisense #13: 5′-AAGGAGTTATATCCAGATTTTCCTGTCTC-3′, mROLP Sense #13: 5′-AAAAAATCTGGATATAACTCCCCTGTCTC-3′. The oligos used to synthesize Epb4.1/3 silencers were: mEpb4.1/3 #1 Antisense 5′-AAGCTCTCTCAGAGATCATCCCCTGTCTC-3′ and mEpb4.1/3 #1 Sense 5′-AAGGATGATCTCTGAGAGAGCCCTGTCTC-3′; mEpb4.1/3 #2 Antisense 5′-AAG GAGTGGAGATTATGTTAGCCTGTCTC-3′ and mEpb4.1/3 #2 Sense 5′-AACTAACATAATCTC CACTCCCCTGTCTC-3′; mEpb4.1/3 #3 Antisense 5′-AAGGAGGTGCCCGTAGTCCACCCT GTCTC-3′ and mEpb4.1/3 #3 Sense 5′ AAGTGGACTACGGGCACCTCCCCTGTCTC-3′. The three double strand siRNAs were transfected in NIH 3T3 cells at different concentrations (0.1, 1, 10, 100 nM) by using Effectene Transfection Reagent (Qiagen) following manufacturer's instructions. ROLP expression was monitored (at mRNA level by real-time RT-PCR and at protein level by western blotting analysis) after 24, 48 and 72 h of siRNA treatment [1].

Murine fibroblast (NIH 3T3) and human neuroblastoma cells (SKNBE2 and SHSY5Y) were grown in a 5% CO₂ incubator at 37 ºC in DMEM medium (Euroclone), containing 10% FBS (Gibco), 4 mM Glutamine, antibiotics. Subconfluent murine fibroblasts (3T3 cells), were transfected with Fugene HD (Roche) according to the manufacturer’s instructions.

Signal Tranduction AntibodyArrayTM (Cat #: HM3000, Hypromatrix, Inc.) was performed according to manufacturer’s instructions.

NIH-3T3 cells were grown overnight on a 6 cm dish and transfected with a specific silencer. After 30 h of silencing the cells were rinsed once with ice-cold PBS pH8, incubated with 1 mM chemical cross-linking dithiobissuccinimidyl dipropionate (DSP, Pierce Chemical Co., Rockford, IL) for 30 minutes at 25 °C and the reactions were quenched by adding 50 mM Tris-HCl, pH 7.5, for 15 minutes. Cells were then scraped, pelleted, and resuspended in 0.1% Triton X-100, 10 mM Tris, pH 7.5, 1 mM EDTA and protease inhibitor cocktail (mini-complete Roche). After 30 minutes on ice, samples were centrifugated at 11,000 g for 15 minutes and the supernatants quantified (Pierce) were used for the immunoprecipitation. Proteins were incubated for 2 hours at 25 °C with primary polyclonal antibody anti-ROLP and 20 mL Protein G-Sepharose, washed 3 times in ice-cold PBS pH 8. Samples were boiled for 5 minutes in Laemmli sample buffer plus 40 mM DTT and loaded on an SDS-PAGE gel.

Protein samples were quantified using a commercial quantification kit (Protein Assay, Bio-Rad) following manifacturer’s instructions. The samples were analyzed by 10% SDS PolyAcrylamide Gel Electrophoresis (SDS-PAGE) and transferred to a nitrocellulose membrane (Whatman, Inc.) or directly stained with Brilliant Blue G-colloidal concentrate (Sigma). The membranes were initially blocked by an incubation of 2 hours in Tris-buffered saline Tween 20 (TBST; 50 mM Tris-HCl, 150 mM NaCl, pH 7.5, 0.05% Tween 20) containing 5% non-fat dried milk. The blots were then incubated for 1 h with the primary antibody against ROLP protein, (see [1] for a detailed description of the antibody features). After washing with TBST membranes were incubated with peroxidase-conjugated anti-rabbit IgGs (A 0545, Sigma)(1:16,000) for 1 h at room temperature. After washing the reactive bands were revealed with ECL (Amersham Biosciences). The densitometric analysis of protein bands was performed taking advantage of ImageJ software system.

In order to identify the biochemical pathway/s in which ROLP is involved we searched for its intracellular molecular interactor/s. By using a polyclonal ROLP-specific antiserum raised against the murine ROLP recombinant protein we co-immunoprecipitated ROLP together with its putative interactor/s in wild type NIH3T3 (hereafter referred to as wt-NIH3T3) as well as in ROLP-silenced NIH3T3 cells (hereafter referred to as siROLP-NIH3T3) [1]. In these experiments molecular interactions were stabilized by chemical cross-linking taking advantage of DSP (dithiobissuccinimidylpropionate), a lipofilic membrane-permeable ester used for both intracellular and intramembrane conjugation. Before performing the co-immunoprecipitation procedure we tested by western blot analysis (using anti-ROLP IgGs) the occurrence of ROLP silencing detecting a strongly decreased amount of ROLP in siROLP-NIH3T3 with respect to the unsilenced wt-NIH3T3 cell lysates (Figure 1A). Cells were then subsequently subjected to co-immunoprecipitation and the proteins were analyzed by SDS PAGE and revealed by colloidal coomassie staining.

A single band of about 87 KDa was observed in wt-NIH3T3 immunoprecipitation product, whereas the same protein signal was approximately three-fold weaker in the siROLP-NIH3T3 sample (149.2 vs 53.2), consistent with the 87 KDa polypeptide being a ROLP interactor (Figure 1B). The 87 KDa protein extracted from the gel was then analyzed by MALDI-TOF mass spectrometry in order to determine its individual identity. Mass spectrometry unambiguously identified the 87 KDa protein as Epb4.1/3, an oncosuppressor protein belonging to the Protein 4.1 superfamily. These molecules are localized at the interface between the cytoskeleton and the plasma membrane, where they play a significant role both in the maintenance of cell structural stability as well as in the transduction to the nucleus of signals triggered by cell shape modifications [8,9]. To strengthen the observation of a possible ROLP/Epb4.1/3 protein-protein interaction we repeated the co-immunoprecipitation in: 1) Epb4.1/3-silenced NIH3T3 cells (siEpb4.1/3-NIH3T3), 2) Epb4.1/3/ROLP-silenced cells (siEpb4.1/3-siROLP-NIH3T3), 3) control unsilenced wt-NIH3T3 cells. Results showed that neither in siEpb4.1/3-NIH3T3, nor in siROLP/siEpb4.1/3-NIH3T3 a protein product was immunoprecipitated by antiROLP IgGs whereas a band at 87 KDa was immunoprecipitated in NIH3T3-unsilenced cells (Figure 1C,D).

Given the possible interaction of ROLP with a protein involved in cell signaling we decided to search for other possible functional partners acting in the same pathway not detected in the NIH3T3 co-immunoprecipitation experiments. To do this we challenged a cell signalling-specific antibody array that exposes 450 spots of cell signaling-associated antibodies with a wt-NIH3T3 cell lysate. In this experiment the proteins bound to ROLP (thus being its putative interactors) are captured by their specific antibodies immobilizing ROLP to specific spots correspondent to its interactors. The subsequent immunodetection with digoxigenin-labelled ROLP-specific IgGs leaded to the staining of a protein-specific signal in the position/s correspondent to the specific interactor/s. Results showed an intense signal correspondent to Dlg4/PSD95 (NP 031890), a PDZ-containing protein that acts in cell adhesion and in cell-cell contact maintenance (Figure 2).

Interestingly in the same experiment we detected a less intense signal in correspondence with few other spots compatible with a weak interaction of ROLP with six other proteins: Integrin-alpha1 (NP_001028400), Rack 1 (CAI35106), p19S kp1 (NP_035673), Rab3 (NP_848805), Rab5 (NP_080163) and Rab11 (NP_059078) (Figure 2 and Table 1). Among these proteins Integrin-alpha 1 is of particular interest due to its key role in cell adhesion and to the intensity of its signal. Altogether the above results keep in line with the possible interaction of ROLP with PSD95 although its potential functional contact with Integrin-alpha1 and/or one of the other five proteins in a more physiological context cannot be excluded a priori by this approach.

As previous experiments suggested the association of ROLP to the prevention of apoptosis we tested its expression in a set of tumor cell lines, in brain and in NIH3T3 cells (a tissue and a cell line known to express ROLP at a high level). In the same samples we also measured the expression of Epb4.1/3, Integrin alpha 1 and PSD95. Results showed that ROLP is actively transcribed in almost all the tumor cell lines (with an expression peak in Neuro2A), in the brain and in wt-NIH3T3 (Figure 3A). To the contrary, (and consistently with its tumor suppressor activity [10]) Epb4.1/3 was found poorly expressed in all the tumor cell lines and actively transcribed by in Neuro2A cells, in brain and in wt-NIH3T3 cells (Figure 3B). Interestingly, PSD95 and Integrin-alpha1 were also highly expressed in Neuro2A, brain and wt-NIH3T3, thus presenting an expression profile similar to the one of Epb4.1/3 and parallel to the expression peaks of ROLP (Figure 3C,D). The coincident cell type-specificity of expression of these proteins is compatible with the occurrence of their biochemical interaction, although the rather ubiquitary expression of ROLP suggests that this protein might have other still unknown cell type-specific partners.

It has been demonstrated that the over-expression of Epb4.1/3 leads to an increased cellular attachment [15], whereas PSD-95 is critical for the formation and the maintenance of synaptic junctions [6]; in addition, PSD-95 play a role as site-specific organizational center for integral membrane proteins and their downstream signaling molecules in the cortical cytoskeleton [16,17]. Considering these notions together with the subcellular localization of Epb4.1/3 and PSD95 at the plasma membrane and the fact that both proteins play an active role in cell signaling and adhesion we decided to test the possible participation of ROLP to these biological processes.

In our experimental approach the cell adhesion capacity of wt-NIH3T3 and siROLP-NIH3T3 cells was tested by a specific assay ([18] modified as described in material and methods). As control in the same experiment wt-NIH3T3 cells were transfected with an unrelated 21nt long siRNA that targets Ex-FABP, a chicken-specific protein (here referred to as siEx-FABP). Results showed that siROLP NIH3T3 cells have a decreased cell adhesion capacity that leads to the detachment of the cells from the substrate. As expected, in the same conditions both wt-NIH3T3 cells and siEx-FABP-NIH3T3 cells maintained their canonical adherence to the substrate (Figure 4A). A quantitative measurement of this phenomenon was obtained repeating the experiment in the same conditions and measuring by MTT [3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide] test the relative amount of viable adherent cells. Again the results evidenced a significantly diminished number of adherent cells in siROLP-NIH3T3 cells (Figure 4B). Altogether these results keep in line with an active participation of ROLP in cell adhesion.