Figure 1.
Experimental workflow for triple SILAC.
(A) The MDA-MB-231 dox- cells were grown in a defined medium, as described in the experimental section, complemented with essential amino acids Arg and Lys, containing naturally occurring atoms (the light medium) or two of their stable isotope counterparts (the medium and heavy media). The medium culture contained arginine (L-Arg 13C6-14N4) and lysine (L-Lys 13C6-15N2) and the heavy culture contained arginine (L-Arg 13C6-15N4) and lysine (L-Lys 13C6-15N2) amino acids. After eight cell divisions to obtain full incorporation of the labeled amino acids into the proteome, cells were then stimulated or not with Lf isoforms. Equal amounts of cells from each condition were combined, creating a single sample that was then subjected to two fractionations. First, subcellular fractionation into cytosolic and nuclear fractions was carried out. In a second fractionation proteins in each subcellular fraction were separated by 1D PAGE. The gel was cut into 20 slices, proteins were digested in the gel slices with trypsin and the resulting peptides extracted from each gel slice were analyzed by reversed-phase nanoscale liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS). The peptides were electrosprayed into the source of a linear ion trap-orbitrap mass spectrometer (LTQ-orbitrap velos). Bioinformatic analyses were assessed using MFPAQ software that processes the results of the Mascot search engine and performs protein quantification. SILAC light/medium/heavy ratios were assessed by MFPAQ for protein quantification. (B) The subcellular fractionation for the SILAC-screen was assessed using marker proteins of known localization. The markers used were GAPDH for the cytosolic fraction and histone H2B for the nuclear fraction.
Table 1.
Validation of SILAC and mass spectrometry results by Western-blotting and qRT-PCR of differentially expressed proteins in response to low doses of both Lf isoforms.
Table 2.
Validation of SILAC and mass spectrometry results by Western-blotting and qRT-PCR of differentially expressed proteins in response to high doses of both Lf isoforms.
Table 3.
ΔLfRE-like sequences found in the promoters of genes regulated by ΔLf and/or hLf.
Figure 2.
Overall evaluation of overexpressed protein identities.
Pie chart representations of the classification by biological processes of the proteins (as identified by nano-LC Orbitrap-MS/MS) found in response to hLf treatment (A) and ΔLf induction (B). Proteins were classified using the DAVID classification system (http://david.abcc.ncifcrf.gov/). All the categories are statistically significant with P- value, 0.01.
Figure 3.
Overall evaluation of downexpressed protein identities.
Histogram representations of the distribution of the downregulated proteins in response to hLf treatment and ΔLf induction according to their molecular functions (A) and biological processes (B). Gene Ontology categorizations were based on information provided by the online resource PANTHER 9.0 classification system (http://www.pantherdb.org/). Panel (C) shows lower level classifications of biological processes and molecular functions for Lf isoforms regulated responsive genes. Gene Ontology lower level categorizations were expressed as percentages: 100% corresponds to metabolic process, cellular process, catalytic activity or binding categorization, respectively. All the categories are statistically significant with P- value, 0.01.
Figure 4.
Both ΔLf and hLf act as transcription factors and target the SelH promoter.
Panels A and B. SelH mRNA overexpression is not cell specific. MDA-MB 231, HeLa, MCF7 and HEK 293 cells were induced with doxycycline (2 µg/mL) or transfected with pcDNA-ΔLf (A). MDA-MB 231, HeLa, MCF7 and HEK 293 cells were transfected with pcDNA-hLf whereas only MDA-MB 231 cells were treated with exogenous hLf (50 µg/mL) (B). The expression pattern of SelH transcripts in MDA-MB-231, HeLa, MCF-7 and HEK-293 cells after treatment was followed by qRT-PCR. Data are normalized to HPRT and are expressed as the fold increase (2−ΔΔCt) under ΔLf (A) or hLf (B) treatment (n = 3). Panel C. HEK 293 cells were cotransfected with pGL3-SelH-Luc construct (50 ng/well) and pcDNA-hLf expression vector (200 ng/well) encoding intracellular hLf or pcDNA-ΔLf expression vector (200 ng/well) encoding ΔLf. 24 h after transfection, cells were lysed and samples were assayed for protein content and luciferase activity. The relative luciferase activity reported is expressed as the fold increase of the ratio of the pGL3 reporter activity to protein content. Values represent the mean ±SD of triplicates from three independent measurements. Panel D. ΔLf and hLf are recruited in vivo on the SelH promoter. HEK 293 cells were transfected with the pcDNA-hLf or the pCMV-3XFLAG-ΔLf. 24 h post transfection, ChIP assays were performed, using an anti-FLAG (M2), an anti-hLf (α-Lf) and anti-rabbit IgG as nonspecific antibody control (IR). As a further control, the assay was performed without binding of an antibody to the protein G plus Sepharose (NIP). The isolated genomic DNA was analyzed by real time PCR using primers that link the ΔLfRE binding site on the SelH promoter. The results were normalized with the levels of ΔLfRE present in the samples (input). Data are expressed as fold enrichment related to null-transfected cells, and are the mean ±SD of triplicates from three independent assays. *p<0.05; **p<0.01; ***p<0.001.
Figure 5.
In vivo recruitment of ΔLf on new target genes, GTF2F2 and UBE2E1.
Panel A. Differential protein expression was confirmed using Western blotting. MDA-MB-231 dox- cells were lysed 24 h after treatment and samples (20 µg of protein) were subjected to SDS-PAGE and immunoblotted with antibodies specific to GTF2F2 and UBE2E1. Histone H2B served as loading control. Panel B. GTF2F2 and UBE2E1 mRNA are upregulated in ΔLf-expressing MDA-MB-231 dox- cells. Cells were either untreated or induced with doxycycline (2 µg/mL) or transfected with pcDNA-ΔLf or treated with exogenous hLf (50 or 500 µg/mL). mRNA content was determined by qRT-PCR. Panel C. HEK 293 cells were transfected with the pCMV-3XFLAG-ΔLf or pcDNA-hLf. 24 h post transfection, a ChIP assay was performed. ChIP product was then amplified by real time PCR using specific primer pairs targeting the ΔLfRE containing fragment of the each targeted promoter. ChIP assays were performed using an anti-FLAG (M2), an anti-hLf (α-Lf) and anti-rabbit IgG as nonspecific antibody control (IR). As a further control, the assay was performed without binding of an antibody to the protein G plus Sepharose (NIP). The isolated genomic DNA was analysed by real time PCR using primers specific for ΔLfRE putative binding site on GTF2F2 and UBE2E1 promoters. The results were normalized with the levels of ΔLfRE present in the samples (input). Data are expressed as fold enrichment related to null-transfected cells, and are the mean ±SD of triplicates from three independent assays. *p<0.05; **p<0.01; ***p<0.001.